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ACCELERATORE DI IMPRESA PER LA VALORIZZAZIONE DELLA RICERCA BIOTECNOLOGICA

BUSINESS ACCELERATOR FOR TECHNOLOGY TRANSFER IN BIOSCIENCE

Servizi delle Piattaforme Tecnologiche Technological Platforms Services


Sommario / Table of Content LE PIATTAFORME TECNOLOGICHE / TECHNOLOGICAL PLATFORMS ........................3 SERVIZI DELLE PIATTAFORME / PLATFORMS SERVICES ............................................4 GENOMICS AND BIOINFORMATICS ............................................................................................................... 5 PROTEOMICS ............................................................................................................................................... 11 CNS CELL MODEL SYSTEMS ......................................................................................................................... 17 STEM CELLS .................................................................................................................................................. 21 MOLECULAR AND CELLULAR IMAGING ....................................................................................................... 25 ANIMAL MODELS PLATFORM AND ANIMAL PATHOLOGY LABORATORY.................................................... 28 GENETIC ENGINEERING OF MODEL PLANT SYSTEMS AND CROPS .............................................................. 36 MICRO- AND NANO-FABRICATION .............................................................................................................. 40 POLYMER THERAPEUTICS ............................................................................................................................ 46 SHARED SERVICES ........................................................................................................................................ 49

OFFERTA FORMATIVA FILARETE/TRAINING PROGRAMS ..........................................50 INSTRUMENT LIST ...........................................................................................................63 IMAGING ...................................................................................................................................................... 68 SPECTROPHOTOMETRY ............................................................................................................................... 82 SEQUENCING ............................................................................................................................................... 86 GENOTYPING ............................................................................................................................................... 88 REAL TIME PCR ............................................................................................................................................ 92 MOLECULAR BIOLOGY IMAGING ................................................................................................................. 94 HISTOLOGY .................................................................................................................................................. 98 FLUORESCENCE MICROSCOPY ................................................................................................................... 107 CYTOFLUORIMETER ................................................................................................................................... 110 SINGLE CELL PATCH CLAMP ELECTROPHYSIOLOGY ................................................................................... 111 PROTEOMICS ............................................................................................................................................. 113 SURFACE FUNCTIONALIZATION ................................................................................................................. 125 THIN FILMS DEPOSITION ........................................................................................................................... 125 OTHER INSTRUMENTS ............................................................................................................................... 129

CONTACT .......................................................................................................................134

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FONDAZIONE FILARETE

Università degli Studi di Milano, Fondazione Cariplo e Intesa Sanpaolo si sono unite per dare vita alla Fondazione Filarete con lo scopo di creare ed accompagnare iniziative imprenditoriali ad elevato contenuto tecnologico nel campo delle bioscienze. La Camera di Commercio di Milano si è recentemente unita all’iniziativa come socio co-fondatore. Fondazione Filarete ha adottato un duplice profilo operativo per fornire alle imprese un supporto multidisciplinare: Scientifico, tecnologico e logistico tramite l’Acceleratore d’Impresa (oltre 6.000 mq); Consulenza finanziaria di business development nonché investimenti in iniziative selezionate, tramite la partecipata Filarete Investimenti (situata all’interno dell’Acceleratore) Il centro nevralgico dell’Acceleratore è costituito dalle 9 Piattaforme Tecnologiche dotate di competenze all’avanguardia ed in grado di svolgere ricerche proprietarie e ricerche/servizi conto terzi.

Università degli Studi di Milano, Fondazione Cariplo and Banca Intesa Sanpaolo have joined to establish Fondazione Filarete with the mission of creating, hosting and nurturing high-tech entrepreneurial initiatives in the field of bioscience. The Chamber of Commerce of Milan has recently joined the initiative as co-founding member Fondazione Filarete has adopted a dual operational profile to support companies with a multi-disciplinary approach: 

Scientific, technological and logistic support through its Business Accelerator (over 6.000 sq.m); Financial advisory, business development services and financing in selected initiatives through its subsidiary company Filarete Investimenti (embedded in the Accelerator).

The core of the Accelerator are 9 state-of-the art technological platforms performing proprietary research and contract services.

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LE PIATTAFORME TECNOLOGICHE / TECHNOLOGICAL PLATFORMS research and contract services.

Il centro nevralgico dell’Acceleratore di Impresa è costituito dalle 9 Piattaforme Tecnologiche, operate e supervisionate da ricercatori e personale scientifico dell’Università degli Studi di Milano ed accessibili a tutte le aziende insediate. Il sistema delle Piattaforme si propone come punto d’incontro tra biotecnologia, genetica, biologia molecolare, imaging, proteomica, nanotecnologie e scienza dei materiali con l’obiettivo di creare una piattaforma integrata di modelli cellulari e animali per la validazione preclinica di molecole rilevanti a livello terapeutico attraverso l’identificazione, la purificazione e la produzione di nuove entità molecolari (vegetali e animali) ed il profiling molecolare delle loro attività in organismi viventi (cellule o animali). L’attività delle Piattaforme è costantemente monitorata da un Comitato Scientifico, composto da esperti provenienti dal mondo accademico, imprenditoriale e istituzionale, presieduto dal Direttore Scientifico della Fondazione Filarete.

The core of Fondazione Filarete is represented by 9 technological platforms managed by cutting-edge research groups of the University of Milano. The different platforms interact to form a unique interdisciplinary and integrated technological core combining genomics, proteomics, cellular and animal models, molecular imaging with nano and micro technology able to provide preclinical validation of therapeutically relevant molecules through the identification purification and production of new molecular entities from plant and animals, for molecular profiling of their activity in living organisms (cells and animals). The activity of the platforms and their effectiveness in supporting technology development and transfer is regularly monitored by the Scientific Advisory Board of Fondazione Filarete chaired by the Scientific Director and composed by experts coming from academia, industry and international institutions.

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SERVIZI DELLE PIATTAFORME / PLATFORMS SERVICES

Le Piattaforme Tecnologiche di Fondazione Filarete mettono in campo tecnologie d’avanguardia e ricercatori di alto livello. I servizi che sono in grado di offrire vengono proposti a enti di ricerca e aziende, siano esse realtà consolidate o imprese in fase di sviluppo. Per far conoscere all’esterno i servizi delle piattaforme è stato preparato questo documento che da un lato illustra le potenzialità della ricerca svolta e dall’altro elenca i servizi offerti attraverso strumentazioni d’eccellenza.

The Technological Platforms of Fondazione Filarete are composed of cutting edge technologies and high profile researchers. The services they are able to offer are proposed to research centers and companies, either mature or in their developing stages. In order for the platforms services to be known outside we have prepared this document that on one side shows the potential of the research done and on the other lists the services through instruments of excellence.

Alla Fondazione Filarete, ci rendiamo conto che le esigenze di ogni cliente sono diverse e ogni progetto è unico. Tutto quello che facciamo ha come fondamento la qualità, la tempestività, il servizio al cliente e l’attenzione al dettaglio.

At Fondazione Filarete, we understand that every customers’ need are different and every project is unique. And underlying everything we do is a proven commitment to quality, timeliness, customer service and unparalleled attention to detail.

Per ulteriori informazioni sui servizi offerti dalle piattaforme si prega di contattare:

For further information on our services please contact:

services@fondazionefilarete.com

services@fondazionefilarete.com

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GENOMICS AND BIOINFORMATICS REFERENT SCIENTISTS Prof. Fabio Macciardi: Biomedical Sciences and Technologies Department, Università degli Studi di Milano (fabio.macciardi@unimi.it) Prof. Daniele Cusi: Biomedical Sciences and Technologies Department, Università degli Studi di Milano (daniele.cusi@unimi.it) Dott. Giulio Pavesi: Biomolecular Sciencies and and Biotechnology, Università degli Studi di Milano (giulio.pavesi@unimi.it)

TO ORDER/FOR INFORMATION services@fondazionefilarete.com

INTRODUCTION The Genomics Platform, together with the Spin Off KOS, makes use of last generation technologies for genotyping, gene expression analysis and sequencing that allow for production of high-density and high-speed genetic and genomic data. Biological data generation is supported by data analysis through methods ranging from statistical genetics to bioinformatics to in silico reconstruction of molecular pathways to the definition of the pathology model. The Genomic Platform is focused on humans, animals and plants. For humans the goal is to analyze and dissect from a genetic point of view complex phenotypes and the response to drugs. For animals and plants the goal is to correlate genotype and specific phenotype characteristics in order to improve the genetic selection. The Platform has at its disposal 2 Illumina genomic platforms (a 500GSX BeadArray and the new iScan2) with a capacity up to 1.5 billion genotypes per month. The post genomic analysis of the results can provide scientists, breeders and associations with a useful tool to design efficient selection plans based on genotype.

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SERVICE FEES SERVICES

GENOTYPING: HUMANS: CHIP 1M-DUO + SERVICE PER BATCH UP TO 48 SAMPLES CHIP 1M-DUO + SERVICE PER BATCH FROM 49 TO 96 SAMPLES CHIP 1M-QUAD + SERVICE PER BATCH UP TO 96 SAMPLES CHIP 1M-QUAD + SERVICE PER BATCH FROM 97 TO 192 SAMPLES CHIP 660K-QUAD + SERVICE PER BATCH UP TO 96 SAMPLES CHIP 660K-QUAD + SERVICE PER BATCH FROM 97 TO 192 SAMPLES ANIMALS: CHIP 60K-PORCINE + SERVICE PER BATCH UP TO 192 SAMPLES CHIP 60K-PORCINE + SERVICE PER BATCH FROM 193 TO 384 SAMPLES CHIP 60K-PORCINE + SERVICE PER BATCH FROM 384 TO 576 SAMPLES CHIP 50K-BOVINE + SERVICE PER BATCH UP TO 192 SAMPLES CHIP 50K-BOVINE + SERVICE PER BATCH FROM 193 TO 384 SAMPLES CHIP 50K-BOVINE + SERVICE PER BATCH FROM 289 TO 576 SAMPLES GOLDEN GATE: CHIP 384SNPs + SERVICE PER BATCH UP TO 96 SAMPLES CHIP 768SNPs + SERVICE PER BATCH UP TO 96 SAMPLES CHIP 1536SNPs + SERVICE PER BATCH UP TO 96 SAMPLES CHIP 3074SNPs + SERVICE PER BATCH UP TO 96 SAMPLES HUMAN METHYLATION 27 B. C. + SERVICE PER BATCH UP TO 96 SAMPLES MANUAL CLUSTERING 1530 SNP FIXED FEE

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SERVICES SEQUENCING: SINGLE READ WITH 1 FLOW CELL (12.5 GIGA BASES) + SERVICE SINGLE READ WITH 2 FLOW CELL (25 GIGA BASES) + SERVICE SINGLE READ WITH 3 FLOW CELL (37.5 GIGA BASES) + SERVICE SINGLE READ WITH 4 FLOW CELL (50 GIGA BASES) + SERVICE SINGLE READ WITH 5 FLOW CELL (62.5 GIGA BASES) + SERVICE SINGLE READ WITH 10 FLOW CELL (125 GIGA BASES) + SERVICE SINGLE READ WITH 15 FLOW CELL (187.5 GIGA BASES) + SERVICE SINGLE READ WITH 20 FLOW CELL (250 GIGA BASES)+ SERVICE PAIRED END WITH 1 FLOW CELL (25 GIGA BASES) + SERVICE PAIRED END WITH 2 FLOW CELL (50 GIGA BASES) + SERVICE PAIRED END WITH 3 FLOW CELL (75 GIGA BASES) + SERVICE PAIRED END WITH 4 FLOW CELL (100 GIGA BASES) + SERVICE PAIRED END WITH 5 FLOW CELL (125 GIGA BASES) + SERVICE PAIRED END WITH 6 FLOW CELL (150 GIGA BASES) + SERVICE PAIRED END WITH 7 FLOW CELL (175 GIGA BASES) + SERVICE PAIRED END WITH 8 FLOW CELL (200 GIGA BASES) + SERVICE GENE EXPRESSION: Call for prices OTHER SERVICES: QUALITY CONTROL X 48 SAMPLES DNA EXTRACTION FROM LIOPHILIZED (PER SAMPLE) DNA EXTRACTION FROM WHOLE BLOOD (PER SAMPLE) DNA EXTRACTION FROM SEMEN (PER SAMPLE) DNA NORMALIZATION (96 SAMPLES) MICROSATELLITES (EXCLUDING FLUORESCINATED PRIMERS) PCR (96 SAMPLES) PCR (48 SAMPLES) ELECTROPHORESIS (DAY-1 UP TO 192 SAMPLES) ELECTROPHORESIS (DAY-2 PER SAMPLE) SETTING FOR EACH MARKER ANALYSIS (UP TO 192 SAMPLES) Tecan Freedom EVO 75 liquid handler (1 HOUR)

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Applied Biosystems 7500 Real-Time PCR System (1 HOUR) Agilent 2100 Bioanalyzer for electrophoresis (1 HOUR) Spectrophotometer Tecan CM Infinite速 M 200 NanoQuant (1 HOUR) DATA TRANSMISSION Less than 15 GigaByte via ftp Up to 500 Giga via shipping of 1 Portable HD USB drive

PLATFORM CAPABILITIES Sequencing: Identification of genetic variants associated with a disease phenotype. lllumina Genome Analyzer allows the sequencing of an entire genome, as well as small to medium to large candidate regions, enabling 1) discovery and confirmation of SNPs 2) identification of chromosomal rearrangements, including Copy Number Variations (CNVs) 3) mapping of break points 4) detection of rare variants.

Gene expression Gene expression studies and transcriptomics enable a new scale of biological experimentation, opening doors to higher-confidence target discovery, disease classification, and pathway studies.

Biostatistics and bioinformatics Genetic data analysis is performed through new and powerful statistic software for data mining and management. The development of new software and protocols is finalized to solve the problems connected with genetic analysis: statistical power, replication of results, sample stratification, gene-annotation and definition of the molecular pathways involved in the etiology of the disease. Bioinformatics tools are used to perform genomic analysis through multi-variate techniques, machine learning techniques, etc, aimed at setting up epidemiological models of complex genetic traits and building complex diseases models.

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Epigenetics Epigenetics refers to changes in gene expression that are stable between cell divisions, and sometimes between generations, but do not involve changes in the underlying DNA sequence of the organism. DNA methylation is an epigenetic modification known to have widespread effects on gene expression and the dysregulation of methylation has been linked to the etiology of common diseases. The technological development of epigenetic studies is still at the beginning except for DNA methylation for which a sensitive detection assay for profiling methylation status at single-site resolution is available. Pharmacogenomics and pharmacogenetics The goal is to develop an algorithm that discriminates responders from non-responders to the pharmacological treatment with the minimal set of significant SNPs. It is in fact highly desirable, clinically and economically, to establish models to distinguish responders from non-responders and to predict possible outcomes of treatments.

EXAMPLES OF ONGOING PROJECTS Definition of a comprehensive genetic epidemiological model of complex traits like Essential Hypertension (EH) and intermediate phenotypes of hypertension dependent/associated Target Organ Damages (TOD). Development of a primary care diagnostic Lab-on-Chip (LOC) platform based on microarrays of human leukocyte antigens (HLA) typing. The combination of LOC technologies with genomic microarrays of HLA typing will provide a state-of-the-art diagnosis at primary care level, concerning multiple sclerosis and rheumatoid arthritis, which are linked with the HLA CLASS I and II genes. Massive SNP genotyping for cattle (4000) and chicken (2000) in order to map loci of large effect at high resolution using GWA and LDLA methods. The project will enhance radically our capability to identify and utilize the most important causative polymorphisms Mapping genetic loci affecting milk quality traits in dairy cattle populations Identification of genes significantly associated to Schizophrenia or a differential brain activation in areas that have been previously reported to be deregulated in schizophrenia. Identification of copy number variants (CNVs) associated with several complex disorders, like Schizophrenia Longitudinal clinical, cognitive, biomarker and imaging data are used to search for genes that are associated with conversion from normal to mild cognitive impairment (MCI) and from MCI to Alzheimer’s Disease (AD)

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REFERENCES Macciardi F, Cusi D. Context dependency of the salt intake: left ventricular hypertrophy connection. J Hypertens. 25(8):1569-72 (2007) Barlassina C, Dal Fiume C, et al Common genetic variants and haplotypes in renal CLCNKA gene are associated to salt-sensitive hypertension. Hum Mol Genet. 1;16(13):1630-8 (2007) T.Castrignanò, M.D'Antonio,et al ASPicDB: A database resource for alternative splicing analysis. Bioinformatics, 24(10), 1300-1304, (2008) G. Pavesi, F.Zambelli, C.Caggese, G.Pesole. Exalign: a new method for comparative analysis of exon-intron gene structures Nucleic Acids Research 36(8), 47 (2008) F.Zambelli, G.Pesole, G.Pavesi. Pscan: finding over-represented transcription factor binding site motifs in sequences from coregulated or co-expressed genes. Nucleic Acids Res. 2009 May 31. [Epub ahead of print] Chio A, Schymick JC, Restagno G, et al. A two-stage genome-wide association study of sporadic amyotrophic lateral sclerosis Human Molecular Genetics 18 (8) 1524-1532 (2009) Potkin SG, Turner JA, Fallon JA, et al. Gene discovery through imaging genetics: identification of two novel genes associated with schizophrenia Source: Schizophrenia Bulletin 35, 194-194 (2009)

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PROTEOMICS REFERENT SCIENTISTS Prof. Gabriella Tedeschi: Animal Pathology, Hygiene and Public Veterinary Health Department, UniversitĂ degli Studi di Milano (gabriella.tedeschi@unimi.it)

TO ORDER/FOR INFORMATION services@fondazionefilarete.com

INTRODUCTION The Proteomics Platform provides expertise in proteomic sample preparation, protein separation by electrophoretic and chromatographic methods, mass spectrometry analyses, western blot analyses, qualitative and quantitative differential analysis of protein content in biological samples. By monitoring the protein expression profiles and post-translational modifications of proteomic samples, the Platform is able to evaluate the effect of new drugs/molecules on the proteome.

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SERVICE FEES Electrophoresis and blotting SERVICE 1 DE mini gel stained by Blue Coomassie + scan 1 DE mini gel stained by Silver + scan 1 DE mini gel stained by fluorescence staining + scan 2 DE mini gel stained by Blue Coomassie + scan 2 DE mini gel stained by Silver + scan 2 DE mini gel stained by fluorescence staining +scan 2 DE gel (18 cm x 18 cm) stained by Blue Coomassie + scan 2 DE gel (18 cm x 18 cm) stained by Silver + scan 2 DE gel (18 cm x 18 cm) stained by fluorescence staining + scan Blotting of 1 mini gel on PVDF/nitrocellulose Blotting of 1 gel (15 cm x 15 cm ) on PVDF/nitrocellulose Western blotting of mini gel + detection (antibodies excluded) Western blotting of gel (15 cm x 15 cm) + detection (antibodies excluded) Computerized gel analysis and comparison among different gels (per hour)

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Mass spectrometry with MALDI SERVICE

Purity control

Determination of the molecular mass of peptides and proteins by MALDI-TOF

Identification of proteins by MALDI mapping. Excission of protein band(s) or spots, in situ digestion, MALDI/MS analysis of the peptide mixture, Database Search.

Analysis of Glycoproteins. Protein digestion, enzymatic stripping of glycoforms of a pure small protein, MALDI/MS analysis of glycoforms.

Characterization of synthetic oligonucleotides. Automated MALDI/MS analysis of synthetic oligonucleotides

N-terminal sequencing SERVICE

Preparation of sample for N-terminal sequencing (mini 1DE + PVDF blotting)

N-terminal sequencing

Separation of peptides for N-terminal sequencing. Denaturation, Reduction, Alkylation and Enzymatic hydrolysis of a pure protein, RP-HPLC fractionation of peptide mixtures

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Other analyses SERVICE

Aminoacid analysis of biological fluids and proteins after hydrolysis (per analysis)

Reduction and derivatization of cystein residues (per sample)

Affinity chromatography with antibodies/proteins

Mass spectrometry with ESI* SERVICE Purity control LCMS analysis of proteins. Separation of proteins by HPLC and direct determination of their molecular masses Peptide sequencing by tandem MS. Separation of peptides by HPLC or direct analysis of purified samples, fragmentation experiments by MSMS tecniques. (per peptide)

Analysis of metabolytes by LCMS including sample pre-treatment

Identification of proteins by LCMSMS. Excission of protein band(s) or spots, in situ digestion, LCMSMS analysis of the peptide-mixture, Database Search.

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De-novo sequencing by LCMSMS of a peptide in solution

Analysis of post-translational modifications

Instrument rental (per hour)

* To be offered shortly

WARNINGS: We do not accept samples that constitute biohazardous material

PLATFORM CAPABILITIES The following services can be activated under specific request: Data analysis and advice Evaluation of enzymatic activity Studies of limited proteolysis, identification of structural domains Localization of S-S bridges Valuation of the effect of a molecule/factor (drug, growth factor, transgenic organism) on a proteomic pattern Identification of antigens for the production of vaccines (SERPA approach) Identification of biomarkers Complete characterization of the proteome of a biological sample Metabolic profile (metabolites, drugs, xenobiotics) Characterization of unknown components of proteic nature in complex mixtures (food) Development of new protocols or new materials for proteomics (matrices,‌) Classification of microrganisms, identification of microrganisms in biological samples including food.*

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EXAMPLES OF ONGOING PROJECTS Functional meaning of protein nitration in physiological conditions and in neurodegeneration Functional meaning and analysis of protein phosphorilation in neurodegeneration Development of nanomaterials for the enrichment of nitrated proteins

REFERENCES Tedeschi G, Taverna F, Negri A, et al. Serological proteome analysis of Staphylococcus aureus isolated from sub-clinical mastitis Veterinary Microbiology 134, 388-391 (2009) P. Coccetti, et al. The CK2 phosphorylation of catalytic domain of Cdc34 modulates its activity at the G1 to S transition in Saccharomyces cerevisiae. Cell Cycle 10, 1-12 (2008) M. Bollati et al. Recognition of RNA Cap in the Wesselsbron Virus NS5 Methyltransferase Domain: Implications for RNA-Capping Mechanisms in Flavivirus J. Mol. Biol. 385, 140-152 (2009) S. Nonnis et al. Tau is endogenously nitrated in mouse brain: identification of a tyrosine residue modified in vivo by NO. Neurochem. Res. 33, 518-525 (2008) I. Marinoni, et al. Characterization of L-aspartate oxidase and quinolinate synthase from Bacillus subtilis FEBS JOURNAL 275, 5090-5107 (2008) C. Pozzi et al. Study of subcellular localization and proteolysis of ataxin-3 Neurobiol. Disease 30 90-200 (2008) G. Cappelletti et al. Protein tyrosine nitration is associated with cold- and drug-resistanct microtubules in neuronal-like PC12 cells. Neurosci Lett 401, 159-164 (2006)

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CNS CELL MODEL SYSTEMS REFERENT SCIENTISTS Prof. Michela Matteoli: Pharmacology, Chemotherapy and Medical Toxicology Department, UniversitĂ degli Studi di Milano, (michela.matteoli@unimi.it)

TO ORDER/FOR INFORMATION services@fondazionefilarete.com

INTRODUCTION The CNS Cell Platform, in collaboration with the Spin Off NeuroZone, is able to isolate and cultivate several cell types from the CNS. The combination of cell culture to a variety of tools for cell adhesion and drug delivery puts the platform at the forefront of drug discovery. The CNS Cell Platform provides a number of biochemical, imaging and electrophysiological assays to study the response of cells to drug candidates.

SERVICE FEES SERVICE Short, 1-day, training in primary CNS cultures (per person) Long, 4-days, training in primary CNS cultures (per person) Cell fractions (growth cone preparations, synaptosomes, subcellular fractions) and analysis of compound activity (per hour, excluding animals) Western blotting of mini gel + detection (antibodies excluded) Cell preparations (primary neurons, astrocytes, from different brain areas, from different animal models) (per hour, excluding animals) Transfections (per hour, excluding DNA)

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Patch clamp (per hour) Single cell calcium/sodium imaging (per hour) Synaptic vesicles recycling assay - neurons (excluding cell preparation, antibodies and drugs) Immunocytochemistry on primary culture (excluding cell preparation, antibodies and drugs) Data analysis (per hour) Without operator Fluorescence microscopy (per hour)

With operator

BTX ECM 830 Square- wave electroporator (per hour)

PLATFORM CAPABILITIES The following services can be activated under specific request: identification of factors facilitating growth and survival of CNS cells (neurons, astrocytes, microglia); setting and production of purified primary brain cultures at different developmental stages in ready-to-use formats (pure primary cultures of astrocytes, neurons and microglia and mixed co-cultures) combined with application of multidisciplinary assays for development of drugs acting on the CNS; integration of biocompatible polymers and nanostructured substrates with CNS cells for tissue engineering, development of innovative solutions for the delivery of drugs acting on cell-to-cell communication in the CNS.

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EXAMPLES OF ONGOING PROJECTS Dissection of neuroinflammatory processes by reproducing inflammatory cascades “in vitro� by means of microfluidic systems. Two new and complementary technologies (brain chips and brain probes) are developed in order to identify inflammatory intermediaries and the sequence of events that cause neuronal damage.

REFERENCES Bianco F, Pravettoni E et al Astrocyte-Derived ATP Induces Vesicle Shedding and IL-1{beta} Release from Microglia. J. Immunol. 174, 7268-77 (2005) Verderio C., Pozzi D. et al Novel role of SNAP-25 in the modulation of neuronal calcium dynamics. Neuron 41, 599-610 (2004) Verderio C, Bianco F et al Cross talk between vestibular neurons and schwann cells mediates BDNF release and neuronal regeneration. Brain Cell Biology 35(2-3), 187-201 (2007) Pozzi D, Condliffe S, Bozzi Y, et al Activity-dependent phosphorylation of Ser187 is required for SNAP-25-negative modulation of neuronal voltage-gated calcium channels. Proc Natl Acad Sci U S A. 105(1), 323-8 (2008) Lovchik RD, Bianco F., Matteoli M et al Controlled deposition of cells in sealed microfluidics using flow velocity boundaries. Lab on Chip 9(10):1395-402 (2009) E. Menna, et al. Eps8 Regulates Axonal Filopodia in Hippocampal Neurons in Response to Brain-Derived Neurotrophic Factor (BDNF), PLOS BIOLOGY 7 (6), Article Number: e1000138 (2009) Lovchik RD, Tonna N, Bianco F, Matteoli M, Delamarche E. Overflow Microfuidic Networks: from Open Cell Cultures to Closed Microfluidics JACS submitted Lovchik RD, Tonna N, Bianco F, Matteoli M, Delamarche E. A microfluidic device for investigating biomolecular cascades between cell populations Lab Chip submitted

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STEM CELLS REFERENT SCIENTIST Prof. Yvan Torrente: Neurologic Sciences Department, UniversitĂ degli Studi di Milano (yvan.torrente@unimi.it)

TO ORDER/FOR INFORMATION services@fondazionefilarete.com

INTRODUCTION The Stem Cells Platform is able to grow several human stem cell lines and possesses patents for stem cell culture on both serum-based and serum-free media. The facility aims at developing a unique know-how for the culture of human stem-cells and eventually for full scale production of clinical-grade human stem cells.

SERVICE FEES SERVICE Short, 1-day, training in cell cultures (per person)* Long, 4-days, training in cell cultures (per person) * 2 days training in stem cells (per person)* 2-days training in primary cultures (per person)* Growth medium for AC133 stem cells from blood (100 ml) Growth medium for AC133 stem cells from muscle (100 ml)

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Differentiation medium for AC133 stem cells from blood (100 ml) Differentiation medium for AC133 stem cells from muscle (100 ml) Cytofluorimetry (excluding reagents) (per hour) ELISA tests (excluding reagents) (per test) Karyotype (per test) Inverted Microscope with LED Illumination Leica DM IL LED (per hour) * max. 8 trainees

PLATFORM CAPABILITIES The following services can be activated under specific request: Cell delivery: human and mouse adult stem cells of different tissue origin (peripheral blood, muscle, bone marrow, neural, mesenchymal) Specific stem cell populations isolated from pathological tissue o from dystrophic muscles (Duchenne muscular dystrophy, Becker muscular dystrophy, shoulder joint) o from brain tumors like glioblastoma multiforme The platform routinely delivers the following cell types:  human CD133+ stem cells isolated from blood  human CD133+ stem cells isolated from muscle  human mononuclear  human and mouse myoblasts (wild type and dystrophic mdx and scid/mdx mice) from primary cultures  human and mouse fibroblasts from primary cultures  mouse C2C12 myoblast lines  mouse 3T3 fibroblast lines  cancer stem cell from human glioblastoma  clones obtained from cancer stem cells of human glioblastoma

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Validation of muscular regeneration and migratory abilities of human stem cells in an immunocompromised dystrophic animal model.

Staminality tests: self-renewal: percentage of primary colonies able to originate secondary colonies Clonogenic ability Differentiative capabilities: endothelial, hematopoietic, muscular Cytogenetic tests: up-regulation of oncogenes telomer length telomerasic activity

EXAMPLES OF ONGOING PROJECTS Optimization of stem cell therapy for disease of epithelia and skeletal muscle through combined basic and applied research Amplification of human myogenic stem cells in clinical conditions

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REFERENCES Colleoni F, Torrente Y. The new challange of stem cell: brain tumor therapy. Cancer Letters (2008) Meregalli M, Farini A, Torrente Y. Combining stem cells and exon skipping strategy to treat muscular dystrophy. Expert Opin. Biol Ther 8(8), 1051-61 (2008)

Marchesi C, et al. Correlation of circulanting CD133+ progenitor subclasses with a Mild phenotype in Duchenne Muscular dystrophy patients. PLoS ONE 3 (5) (2008) Cassano M, et al. Magic-Factor 1, a Partial Agonist of Met, Induces Muscle Hypertrophy by Protecting Myogenic Progenitors from Apoptosis PLoS ONE 3 (9) (2008) Marchesi C et al. Skin-derived stem cells transplanted into resorbable guides provide functional nerve regeneration after sciatic nerve resection. Glia 55(4), 425-38 (2007) Torrente Y et al. Autologous Transplantation of Muscle-Derived CD133+ Stem Cells in Duchenne Muscle Patients Cell Transpl. 16(1), 1-100 (2007) Torrente Y Restoration of human dystrophin following transplantation of exon-skipping-engineered DMD patient stem cells into dystrophic mice. Cell Stem Cell 1, 1-13 (2007)

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MOLECULAR AND CELLULAR IMAGING REFERENT SCIENTISTS Dott. Maura Francolini: Pharmacology, Chemotherapy and Medical Toxicology Department, UniversitĂ degli Studi di Milano (maura.francolini@unimi.it) Dott. Simona Rodighiero: Fondazione Filarete (simona.rodighiero@fondazionefilarete.com)

TO ORDER/FOR INFORMATION services@fondazionefilarete.com

INTRODUCTION The Imaging Platform applies optical methods to the study of molecule localization and interaction in subcellular structures, living cells and tissues. The platform provides bioinformatic support for data storage and image analysis.

SERVICE FEES SERVICE

Confocal microscope Leica TCS SP5 AOBS with temperature and CO2 control; perfusion chamber for round coverslips (1 hour)

without operator with operator without operator

Confocal microscope Leica TCS SPE UV (1 hour) with operator Training for use of the confocal microscopes (per person)

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Cell culture and treatments (transfection) (1 hour) Sample preparation for Electron Microscopy (scanning or transmission) (per sample)

Cut sectioning for Transmission Electron Microscopy (T.E.M.) with Leica Ultracut UC6 (1 hour)

without operator with operator

Scanning electron microscope Zeiss Sigma Gemini with STEM detection system for observation of samples in the transmission mode (T.E.M.) (1 hour) Data analysis (1 hour)

PLATFORM CAPABILITIES The following services can be activated under specific request: Confocal microscopy on fixed samples. Labeling and detection of target molecules (proteins, lipids,‌) using multicolor fluorescence. Examples: o Subcellular localization o Expression levels Confocal microscopy on live cells. o Analysis of molecules interaction in vivo with high spatio-temporal resolution thanks to the application of Fluorescence Resonance Energy Transfer (FRET) o Analysis of molecules fate and behaviour in cells in vivo through Fluorescence Recovery After Photobleaching (FRAP) o Time lapse imaging (for example: internalization of plasma membrane domains, functional screening of anion transporters,‌) Electron Microscopy studies, Correlative Microscopy Digital image analysis (deconvolution, 3D rendering) User assistance (project planning, sample preparation, staining, microscope selection and use, image processing, presentation, data transfer and storage) Collaboration with industrial partners (testing microscopy equipment and imaging software for third parties)

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EXAMPLES OF ONGOING PROJECTS Functional screening of genetic mutations of Pendrin. We take advantage of EYFP protein sensitivity to halides in transiently transfected cells expressing both EYFP and Pendrin (WT or mutated) proteins. Quantitative analyses of EYFP fluorescence variation following changes in extracellular halides can be related to the overexpressed anionic exchanger. FRET study of the dynamic interaction between ICln and 4.1R in the context of Regulatory Volume Decrease (RVD), the process adopted by living cells to counteract osmotic swelling. The dynamic interaction between the two proteins is investigated by the use of FRET.

REFERENCES Moscatelli A, Ciampolini F, Rodighiero S, Onelli E, Cresti M, Prescianotto-Baschong C. Distinct endocytic pathways identified in tobacco pollen tubes using charged nanogold. J. Cell Sci. Nov 1;120(Pt 21), 3804-19. Snapp E.L., Hedge R.S., Francolini M., Lombardo F., Colombo S., Pedrazzini E., Borgese N. and Lippincott-Schwartz J. ,Formation of stacked ER cisternae by low affinity protein interactions J. Cell Biol.163 (2), 257-269 (2003) Ronchi P., Colombo S., Francolini M. and Borgese N. Transmembrane domain dependent partitioning of membrane proteins within the endoplasmic reticulum�. J. Cell Biol. 181(1), 105-118 (2008) Rodighiero S, Bazzini C, Ritter M, Fßrst J, Botta G, Meyer G, Paulmichl M. Fixation, mounting and sealing with nail polish of cell specimens lead to incorrect FRET measurements using acceptor photobleaching. Cell Physiol Biochem. 21(5-6), 489-98 (2008) Dossena S, Rodighiero S, et al. Functional characterization of wild-type and mutated pendrin (SLC26A4), the anin transporter involved in Pendred syndrome. J Mol Endocrinol. 2009 Jul 16. [Epub ahead of print] Pera A, Dossena S, Rodighiero S et al. Functional assessment of allelic variants in the SLC26A4 gene involved in Pendred-Syndrome and non-syndromic EVA. PNAS 105(47), 18608-13 (2008) Dossena S, Rodighiero et al. Fast fluorometric method for measuring pendrin (SLC26A4) Cl-/I- transport activity. Cell Physiol Biochem 18(1-3), 67-74 (2006) M. FrancoliniM., et al. Glutamatergic reinnervation and assembly of glutamatergic synapses in adult rat skeletal muscle occurs at cholinergic endplates J. Neuropathol. Exp. Neurol. In press

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ANIMAL MODELS PLATFORM AND ANIMAL PATHOLOGY LABORATORY REFERENT SCIENTIST Prof. Adriana Maggi – Animal Engineering Lab: Director of the Center of Excellence on Neurodegenerative Diseases, Università degli Studi di Milano (adriana.maggi@unimi.it) Prof. Eugenio Scanziani - Mouse & Animal Pathology Lab: Dean of the School of Veterinary Medicine, Università degli Studi di Milano (eugenio.scanziani@unimi.it)

TO ORDER/FOR INFORMATION services@fondazionefilarete.com

INTRODUCTION The Animal Pathology laboratory provides in depth analysis of the phenotype of animal models. Its expertise relies on the characterization of histological and immunohistochemical samples. The platform aims to offer its services to basic research groups and pharmaceutical companies for the study of drug action in complex model systems and for the generation of models in which preclinical and clinical phases of drug research can proceed as a continuum thanks to the use of markers of drug action and methodologies (imaging) which can be used in small animals as well as in humans.

SERVICE FEES ANIMAL PATHOLOGY SERVICES Necropsy For single sample Necropsy with sampling and reporting (mouse) Necropsy + Histology (mouse) (1 tissue; for additional tissues see below) Necropsy with sampling and reporting (rat)

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Necropsy + Histology (rat) (1 tissue; for additional tissues see below)

2-5 samples Necropsy with sampling and reporting (mouse) Necropsy + Histology (mouse) (1 tissue; for additional tissues see below) Necropsy with sampling and reporting (rat) Necropsy + Histology (rat) (1 tissue; for additional tissues see below)

Histology For single sample Trimming, embedding sectioning, H&E staining standard (trimming, embedding, sectioning, H&E staining, reading and reporting) for block increment special sectioning and staining

2-5 samples Trimming, embedding sectioning, H&E staining standard (trimming, embedding, sectioning, H&E staining, reading and reporting) for block increment special sectioning and staining

6-15 samples Trimming, embedding sectioning, H&E staining

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standard (trimming, embedding, sectioning, H&E staining, reading and reporting) for block increment special sectioning and staining

> 15 samples Trimming, embedding sectioning, H&E staining standard (trimming, embedding, sectioning, H&E staining, reading and reporting) for block increment special sectioning and staining

Immunohistochemistry For single sample Staining standard * for block increment

2-5 samples Staining standard * for block increment

6-15 samples Staining standard * for block increment

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> 15 samples Staining standard * for block increment

Automatic Image analysis For 1-10 samples reading and reporting

For 11-30 samples reading and reporting

>30 samples reading and reporting

Image analysis with manual tag For 1-10 samples reading and reporting

For 11-30 samples reading and reporting

>30 samples reading and reporting

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Microphotographs Microphotograph (electronic file) Microphotograph with optimization, symbols and legend (electronic file)

Microbiology tests Bacteriological test Bacterial strain identification Antibiogram

Consultancy Histology: evaluation of a single case. Report including AFIP style description, Morphological diagnosis, Comment, Answers to specific questions Peer-review of pathological evaluation in toxicological studies (per tissue) Consultation at MAPLab of histoslides of didactic interest with corresponding AFIP style description, Morphological Diagnosis and Comment

* = 20% discount if customer supplies primary antibody

ANIMAL MODEL SERVICES Real Time PCR 7900 HT Fast Real Time PCR combines 96 and 384-well plate compatibility also offers optional Fast real-time PCR capability. Combined with TaqMan Genomic assays enable to achieve unprecedented throughput and flexibility, allowing to pursue projects beyond the scope of previous real-time instruments. In their 96 well format these Instruments allow to run 96 samples in less than 2 hours. It is possible to update to 384 well plate format or to the Micro Fluid card format. Taq Man Assay (assay design samples quality control, reaction, analyses)

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Design Taq Man Assay (assay design samples quality control, reaction, analyses)

Micro Fluidic Card (assay design samples quality control, reaction, analyses) cDNA RNA extraction

Retro-transcription

Maxi – Plasmid DNA Preparation Kit qiagen, Plasmid quality Preparation, high purification (transfection in eukaryotic cells 500 ug – 1 mg) Mini – Plasmid DNA Preparation Kit qiagen, Plasmid quality Preparation Competent Cells Competent cells bacterial strains : xl1blue (107) , dh5alpha (106) , HB101 (106), sure (105) Transformation Transformation of plasmid DNA into bacteria cells : xl1blue , dh5alpha , HB101, sure Qualitative/quantitavie RNA analysis with Experion automated electrophoresis system Qualitative/quantitavie PROTEIN analysis with Experion automated electrophoresis system Qualitative/quantitavie DNA analysis with Experion automated electrophoresis system Spectrophotometer Tecan CM Sunrise™ (1 hour) Spectrophotometer Nanodrop ND-1000 (1 hour) Biorad Experion 700-7002 Automated Electrophoresis Station (1 hour) Promega GloMax®-Multi Detection System (1 hour) Upright Microscope Leica DM2500 (1 hour) Manual Inverted Microscope Leica DMI3000 B (1 hour) Real Time PCR rental (per hour) * = for al least 10 items

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PLATFORM CAPABILITIES The following services can be activated under specific request: ANIMAL PATHOLOGY LABORATORY In lab practical technical training Preparation or review of scientific papers or internal reports Preparation or review of oral presentations Organization of courses or scientific meetings Ad hoc training programs for veterinary pathologists

ANIMAL ENGINEERING PLATFORM Modification of models of human diseases for the study of novel therapies by the use of imaging Application of innovative animal models, such as reporter mice, to pharmacological research Application to imaging technologies to the study of drug pharmacokinetics and pharmacodynamics Generation of stem and neural reporter cells Generation of innovative systems of immobilized reporter cells for drug functional screening Generation and testing of innovative systems for drug delivery A practical training in the field of Animal engineering and Imaging

REFERENCES Stell A., Et Al, Multimodality imaging: novel pharmacological applications of reporter systems. Q J Nucl Med Mol Imaging. 51, 127-38 (2007) Stell A., Et Al Cancer modeling: modern imaging application in a generation of novel animal system sto study cancer progression and therapy. Int. J. Biochem Cell Biol. 39, 1288 - 96 (2007) Maggi A., Salvatore M., Molecular Imaging. Q J Nucl Med Mol Imaging. 51, 95 (2007) Stell A., Belcredito S., Ramachandran B., Biserni A., Rando G., Ciana P., Maggi A. Multimodality imaging: novel pharmacological applications of reporter system. Q J Nucl Med Mol Imagin. 51, 127-38 (2007)

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Montani C., Penza M., Jeremic M., Biasiotto G., La Sala G., De Felici M., Ciana P., Maggi A., Di Lorenzo D. Genistein is an efficient estrogen in the whole-body throughout mouse development. Toxicol. Sci. 103, 57 – 67 (2008) Biserni A., Gianessi F., Sciarroni Af. Milazzo Fm., Maggi A., Ciana P. In vivo imaging reveals selective peroxisome proliferator activated receptor modulator activity of the synthetic ligand 3-(1-(4-chlorobenzyl -3-t-butylthio-5-isopropylindol-2-yl)-2,2dimethylpropanoic acid (MK-886). Mol. Pharmacol. 73, 1434 – 43 (2008) Cignarella A., Bolego C., Pelosi V., Meda C., Krust A., Pinna C., Gaion R.M., Vegeto E., Maggi A. District roles of estrogen receptor-α and β in the modulation of vascular inducible NO synthase diabetes. J Pharmacol Exp Ther. Oct 2 (2008) Vegeto E., Benedusi V., Maggi A. Estrogen anti-inflammatory activity in brain: A therapeutic opportunità for menopause and neurodegenerative diseases. Frontiers in Neuroendocrinology 29, 507-19 (2008) Di Lorenzo D., Rando G., Ciana P., Maggi A. Molecular imaging, an innovative methodology for whole body profiling of endocrine disrupter action. Toxicological Sciences 106, 304 – 311 (2008) Stell A., Belcredito S., Ciana P., Maggi A. Molecular imaging provides novel insights on estrogen receptor activity in mouse brain. Molecular Imaging 7, 11 – 21 (2008) Amadasi A., Mozzarelli A., Meda C., Maggi A., Cozzini P. Identification of Xenoestrogens in food additives by an integrated in silico and in vitro approach. Chem. Res. Toxicol. 22, 52 – 63 (2009) Brufani M., et al. Novel Locally Active Estrogens Accelerate Cutaneous Wound Healing. A Preliminary Study. Molecular Pharmaceutics. 6, 543-556 (2009)

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GENETIC ENGINEERING OF MODEL PLANT SYSTEMS AND CROPS REFERENT SCIENTISTS Prof. Chiara Tonelli: Biomolecular Sciences and Biotechnology Department, Università degli Studi di Milano (chiara.tonelli@unimi.it) Dr. Massimo Galbiati: Biomolecular Sciences and Biotechnology Department, Università degli Studi di Milano (massmo.galbiati@unimi.it)

TO ORDER/FOR INFORMATION services@fondazionefilarete.com

INTRODUCTION The Plant models Platform is equipped with a laser microdissector for the isolation of tissues or cells from plant or animal sources. Goals of the platform are the identification and manipulation of genes that improve the resistance of crops to environmental stresses and the production of pharmaceuticals and nutraceuticals in plants.

SERVICE FEES SERVICE Growing plants to obtain tissues of interest Feasibility study with 10 slides: Block preparation Sectioning and slide preparation

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Standard staining (optional)

Leica LMD6000 Laser Microdissection of animal and plant tissues (1 hour)

without operator with operator

DNA/RNA extraction DNA/RNA quantification/quality control

Stereomicroscope Leica M205 FA (1 hour) LI-COR LI-6400XT gas Exchange system (1 hour)

PLATFORM CAPABILITIES The following services can be activated under specific request: Plant transformation Development of promoters and their insertion in vectors for the conditional expression of genes in specific plant cells and tissues Imposition of controlled multiple stresses which mimic the field conditions (To be offered shortly) Identification of genes controlling the tissue/cell-specific adjustments that shape adaptive traits in plants: o Microdissection of tissues/cells of interest o Gene profiling analyses of Laser Microdissected-purified samples o Gene trap screens (Arabidopsis) Modulation of tissue/cell-specific candidate genes in crops for stress tolerance.

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Development of plants containing high levels of antioxidants (anthocyanin and flavonoids). Example: introgress the high-anthocyanin trait into corn varieties suitable for human consumption

EXAMPLES OF ONGOING COLLABORATIONS Large-scale gene trap system for functional genomics studies in Arabidopsis. In collaboration with John Innes Institute, Norwich, U.K. Exploitation of guard cell-specific promoters for water use efficiency and drought tolerance in fruit trees. In collaboration with Huazhong Agricultural University, Whuan, China. Analysis of MYB transcription factors from grape: functional studies and evaluation of potential biotechnological applications. In collaboration with Pontificia Universidad Catholica de Chile.

HIGHLIGHTS The working group has patented a cell-specific promoter for the expression of recombinant proteins in guard cells to decrease water consumption and enhance drought tolerance in Arabidopsis and has licensed it to one of the world’s leading agribusinesses The working group has patented a cell-specific promoter for the expression of recombinant proteins in embryo and has licensed it to one of the world’s leading agribusinesses

REFERENCES M.-C. Toufektsian, M. de Lorgeril, P. Salen, N. Nagy, M. B. Donati, L. Giordano, H.-P. Mock, S. Peterek, K. Petroni, R. Pilu, D. Rotilio, C. Tonelli, J. de Leiris, F. Boucher, C. Martin. Chronic dietary intake of plant-derived anthocyanins protects the rat heart against ischemiareperfusion injury Journal of Nutrition 138(4), 747-752 (2008) Giuntini D, Lazzeri V, Calvenzani V, C. Dall’Asta, G. Galaverna, C. Tonelli, K. Petroni and A. Ranieri. Flavonoid profiling and biosynthetic gene expression in flesh and peel of two tomato genotypes grown under UV-B-depleted conditions during ripening. Journal Of Agricultural And Food Chemistry 56 (14), 5905-5915 (2008)

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R. Pilu, P.Piazza, K. Petroni, A. Ronchi, C. Martin, C.Tonelli pl-bol3, a complex allele of the anthocyanin regulatory pl1 locus that arose in a naturally occurring maize population. Plant Journal 36:510-521 (2003) Cominelli E, Galbiati M, Tonelli C. , Bowler C. Water: the invisible problem Access to fresh water is considered to be a universal and free human right, but dwindling resources and a burgeoning population are increasing its economic value. Embo Reports, 10 (7), 671-676 (2009) Cominelli E, Gusmaroli G, Allegra D, Galbiati M, Wade H.K, Jenkins G.I, Tonelli C Expression analysis of anthocyanin regulatory genes in response to different light qualities in Arabidopsis thaliana. Journal of Plant Physiology 165 (8), 886-894 (2008) E. Cominelli, T. Sala, D. Calvi, G. Gusmaroli, C. Tonelli. Overexpression of the Arabidopsis AtMYB41 gene alters cell expansion and leaf surface permeability. Plant Journal 53, 53-64, 2008. Galbiati M, Simoni L, Pavesi G, , E; Francia, P; Vavasseur, A; Bevan, M; Nelson, T; Tonelli, C Gene trap lines identify Arabidopsis genes expressed in stomatal guard cells. Plant Journal 53 (5), 750-762 (2008) E. Cominelli, M. Galbiati, A. Vavasseur, L. Conti, T. Sala, M. Vuylsteke, N. Leonhardt, S.L. Dellaporta, C. Tonelli. A guard cell-specific MYB transcription factor regulates stomatal movements and plant drought tolerance. Current Biology, 15,1196-1200, 2005.

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MICRO- AND NANO-FABRICATION REFERENT SCIENTISTS Dott.ssa Cristina Lenardi: Department of Molecular Science Applied to Biosystems, UniversitĂ degli Studi di Milano (cristina.lenardi@unimi.it) Prof. Paolo Milani: Director of C.I.Ma.I.Na., UniversitĂ  degli Studi di Milano (paolo.milani@mi.infn.it)

TO ORDER/FOR INFORMATION services@fondazionefilarete.com

INTRODUCTION The Micro- and Nano-Fabrication Platform designs and produces devices aimed at the culture, manipulation and imaging of single cells and cell populations. Micro- and nano- structured custommade surfaces provide control on cell attachment, growth, proliferation and phenotype. Some capabilities are: handling and dispensing of nutrients and chemical stimuli in the liquid phase, displacement and handling of cells and integration of in situ analytical tools including electronic and optical ones.

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SERVICE FEES SERVICE Scanning Electron Microscopy (SEM) (1 hour) Technical Specifications: ZEISS SIGMA (May 2009) - Thermal Field Emission Electron Source Acceleration Voltage: 0,1 – 30 kV Resolution: up to 1.5nm Current: 4pA-40nA Magnification: 12x – 500000x Sample dimensions: up to 125mm x 125mm This instrument guarantees optimal performances also in the visualization of insulating samples like polymers and biological systems.

with operator

Transmission Electron Microscopy (SEM + STEM detector) (1 hour) with operator Technical Specifications: STEM detector Zeiss. Loading capacity: 12 standard TEM grids. Electron microscopy with elemental analysis (SEM + EDX detector) (1 hour) Technical Specifications: with operator Bruker Energy Dispersive X- Ray Spectrometer Quantax 400 able to produce maps of chemical contrast with submicrometric lateral resolution. E-beam lithography (SEM + Elphy Quantum RAITH) (1 hour) Technical Specifications: The Raith Elphy Quantum pattern generator is able to machine surfaces with submicrometric lateral resolution. Machining patterns can be designed with the relative CAD software. Profiles provided in jpeg electronic format can also be handled and impressed on surfaces.

with operator

Sample preparation for Electron Microscopy (scanning or transmission) (per sample) Sample handling for e-beam lithography processes: CAD service, primer and resist deposition and treatments, after exposition lift off processes (1 hour)

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Data handling, analysis and report production (1 hour) without operator Cut sectioning for Transmission Electron Microscopy with Leica Ultracut UC6 (1 hour) with operator Stylus profilometry: measurement of films thickness and roughness (1 hour)

without operator

Technical Specifications: VEECO Dektak 150 with Low-Force head and Advanced Automation Package setup

with operator

without operator Spotters for microarrays (1 hour) with operator Scanner (per slide) Technical Specifications: TECAN Power-Scanner: dual channel 635&532nm, 4 Âľm image scanning, 48 slides autoloader.

without operator with operator

Scanner (batch of 48 slides)

Optical microscopy with fluorescence setup (1 hour) Cleanroom (1 hour) Spin Coating (1 hour) NanoOnSi nanostructured thin films deposition (1 hour) Evaporator (1 hour) Plasma system (1 hour)

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PLATFORM CAPABILITIES The following services can be activated under specific request: Chemical and physical functionalization of materials and surfaces: -

plasma etching and thermal treatments thin films deposition of metals and oxides nanostructured and cluster assembled thin films deposition deposition of thin polymer films by spin-coating

Lithography -

electron beam lithography lift off processes

Design and prototyping of microstructured platforms and devices for biological applications. Available technologies: -

Protein microarrays Integration of in-situ analytical tools, microfluidic devices, electrical contacts in biologicalfriendly environment Integration of nanomaterials on microfabricated platforms

EXAMPLES OF ONGOING COLLABORATIONS Design and production of nanostructured substrates, arrays and microsystems for cell adhesion, growth, manipulation and imaging Development of nanostructured and microfabricated surfaces for cell growth and realization of BioMEMS Study of the differentiation and cytoskeletal protein dynamics of cells cultured on clusterassembled ns-TiOx thin films Development of new enrichment techniques for analysis of nitropeptides Protein-surface interaction microarrays for the high-throughput characterization of protein adsorption on a library of nanostructured biomaterials Localized implantation of metal clusters in polymers and their functionalization Development of imaging protocols with Scanning Transmission Electron Microscopy combined with elemental analysis EDS Development of microdevices for cell cultures

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REFERENCES A. Podestà, G. Tiana, M. Manno, P. Milani, Early events in insulin fibrillization studied by time-lapse atomic force microscopy Biophysical Journal 90, 589 (2005) C, Lenardi, C. Perego, V. Cassina, A. Podestà, A. D'Amico, D. Gualandris, S. Vinati, F. Fiorentini, G. Bongiorno, P. Piseri, F. Vellea Sacchi, P. Milani Adhesion and Proliferation of Fibroblasts on Cluster-Assembled Nanostructured Carbon Films: The Role of Surface Morphology Journal of Nanoscience and Nanotechnology, 6, 3718 (2006) R. Carbone, I. Marangi, A. Zanardi, L. Giorgetti, E. Chierici, G. Berlanda, A Podestà, F. Fiorentini, G. Bongiorno, P. Piseri, P. G. Pelicci, P. Milani Biocompatibility of cluster-assembled nanostructured TiO2 with primary and cancer cells Biomaterials 27, 3221 (2006) K. Wegner, P. Piseri, H. Vahedi Tafreshi, P. Milani Cluster beam deposition: a tool for nanoscale science and technology Journal of Physics D: Applied Physics 39, R439 (2006) R. Carbone, L. Giorgetti, A. Zanardi, I. Marangi, E. Chierici, G. Bongiorno, F. Fiorentini, M. Faretta, P. Piseri, P. G. Pelicci, P. Milani Retroviral microarray-based platform on nanostructured TiO2 for functional genomics and drug discovery Biomaterials 28, 2244 (2007) A. Zanardi, L. Giorgetti, O.A. Botrugno, S. Minucci, P. Milani, P.G. Pelicci, R. Carbone Immunocell-array for molecular dissection of multiple signaling pathways in mammalian cells Molecular and Cellular Proteomics, 6, 939 (2007) M. Indrieri, M. Suardi, A. Podestà, E. Ranucci, P. Ferruti, P. Milani Quantitative investigation by Atomic Force Microscopy of supported phospholipids layers and nanostructures on cholesterol-functionalized glass surfaces Langmuir 24, 7830 (2008) L. Giorgetti, G. Bongiorno, A. Podestà, G. Berlanda, P. Scopelliti, R. Carbone, P. Milani Adsorption and stability of streptavidin on cluster-assembled nanostructured TiOx films Langmuir, 24, 11637 (2008) G. Lia, M. Indrieri, T. Owen-Hughes, L. Finzi, A. Podestà, P. Milani, D. Dunlap ATP-dependent looping of DNA by ISWI Journal of Biophotonics 1, 280 (2008) E. Jacchetti, E. Emilitri, S. Rodighiero, M. Indrieri, A. Gianfelice, C. Lenardi, A. Podesta', E. Ranucci, P. Ferruti, P. Milani Biomimetic poly(amidoamine) hydrogels as synthetic materials for cell culture Journal of Nanobiotechnology, 6, 14 (2008)

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L. Ravagnan, G. Divitini, S. Rebasti, M. Marelli, P. Piseri, P. Milani Poly(methyl methacrylate) - Palladium clusters nanocomposite formation by supersonic cluster beam deposition: a method for microstructured metallization of polymer surfaces Journal of Physics D: Applied Physics 42, 082002 (2009) R. Carbone, M. De Marni, A. Zanardi, S. Vinati, E. Barborini, L. Fornasari, P. Milani Characterization of cluster-assembled nanostructured titanium oxide coatings as substrates for protein arrays Analytical Biochemistry, 394, 7 (2009) A.V. Singh, C. Lenardi, L. Gailite, A. Gianfelice, P. Milani A simple lift-off based patterning method for micro- and nanostructuring of functional substrates for cell colture Journal of Micromechanics and Microengineering, in press

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POLYMER THERAPEUTICS REFERENT SCIENTISTS Dott.ssa Cristina Lenardi: Department of Molecular Science Applied to Biosystems, UniversitĂ degli Studi di Milano (cristina.lenardi@unimi.it) Dott. Federico Martello: Fondazione Filarete (federico.martello@fondazionefilarete.com)

TO ORDER/FOR INFORMATION services@fondazionefilarete.com

INTRODUCTION The Polymer Therapeutics Platform is equipped for chemical synthesis or modifications of organic compounds. In particular, the platform possesses the capability of producing and characterizing a number of tailor-made polymeric substances or performing chemical modification of natural polymers. The expertise of the platform includes synthesis and characterization of a number of polymers for biomedical applications, such as polyurethanes (PU), poly(2hydroxyethylmethacrylate) (PHEMA), poly(N-isopropylacrylamide) (PNIPAAM), polyvinylpirrolidone (PVP), poly(lactic-co-glycolic) acid (PLGA), poly(amidoamine)s (PAA). The physico-chemical properties of all these polymers can be adjusted by introducing chemical functionalities in the polymer backbone or by controlling the polymer molecular weight and the branching degree. Many polymers can also be obtained in a reticulated and insoluble form (i.e. hydrogels).

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SERVICE FEES SERVICE

Polyamides (e.g. nylon 6; 6,6; 6,10) (per gram) Polyacrylates (e.g. PMMA, PHEMA, PNIPAAM, polyacryloylmorpholine) (per gram)

Polyesters (e.g. PET) (per gram)

Silicones (e.g. PDMS) (per gram)

Polyamidoamines (per gram)

PLATFORM CAPABILITIES The following services can be activated under specific request: Assays and chemical analyses (e.g. HPLC, acid-base titrations, UV-Visible spectroscopy, Infrared spectroscopy ATR-FTIR). Identification of active ingredients and organic impurities in the materials. Separation, identification and quantification of each constituent in an unknown material (HPLC, titration, UV-Vis, IR, Nuclear Magnetic Resonance). Analysis of chemical structure by Size Exclusion Chromatography/HPLC, UV-Vis, ATR-FTIR. Analysis of thermal properties by Differential Scanning Calorimetry (DSC), Thermogravimetric Analysis (TGA). Analysis of mechanical properties by Rheometry and Dynamic Mechanical Analysis (DMA). Degradation behaviour of the material under different environmental conditions (stove, SEC, UV-Vis, ATR-FTIR). Biological tests involving the use of cell lines, primary cells, bacteria and fungi can be performed in collaboration with other Platforms of Fondazione Filarete. Moreover, in vivo biological tests can be performed. The Polymer Therapeutics Platform offers also advices to research group and companies for improvement and optimization of polymer synthesis and production process, with emphasis on applications in the biomedical field.

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EXAMPLES OF ONGOING PROJECTS Synthesis of biocompatible and biodegradable hydrogels as scaffolds for tissue regeneration. An interpenetrating polymer network (IPN) with polymer such as PEG, PHEMA, PLGA is used to produce three dimensional scaffolds that induce primary cell growth and proliferation in vivo. Synthesis of tailor-made biocompatible, biodegradable and pH-responsive polymers for drug delivery applications. Different series of functionalized polyamidoamines are synthesized and loaded with several bioactive molecules. These formulations are able to release the loaded molecules in the external environment under specific stimuli (i.e. pH variation).

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SHARED SERVICES

without operator Chemidoc (1 hour) with operator without operator Versadoc (1 hour) with operator

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OFFERTA FORMATIVA FILARETE/ TRAINING PROGRAMS Filarete è impegnata a fornire un’offerta formativa in grado di presentare le tecnologie avanzate in dotazione alla struttura e a mettere a disposizione di terzi le conoscenze acquisite dalle piattaforme tecnologiche nelle aree: Genomica e bioinformatica, modelli cellulari, cellule staminali, imaging cellulare e molecolare, modelli animali, patologia veterinaria, modelli vegetali, micro e nano tecnologie e polimeri. Al momento sono disponibili i seguenti corsi teorici/pratici:

Filarete is committed to provide an educational training programs Filarete is committed to provide training programs to present our advanced technologies and know-how in the following areas: Genomics and Bioinformatics, cell model systems, stem cells, molecular and cellular imaging, animal models and animal pathology laboratory, model plant systems, micro and nano fabrication and polymer. Currently our offer is on the following practical and theoretical courses:

IMAGING Microscopia confocale. Preparazione campioni per microscopia elettronica Confocal microscopy. Sample preparation for Electron microscopy (TEM,STEM) MICRO E NANO TECNOLOGIE (MICRO AND NANO TECHNOLOGIES) Microscopia elettronica. Electron Microscopy STAMINALI (STEM CELL) Training in colture cellulari e cellule staminali. Training in stem cells and primary cultures. MODELLI ANIMALI (ANIMAL MODEL) Imaging molecolare per drug discovery. Molecular imaging for drug discovery. MODELLI CELLULARI (CNS CELL MODEL CNS) Colture cellulari e trasfezione. Colture primarie CNS sistema nervoso centrale Training in primary CNS cultures and transfections PROTEOMICA (PROTEOMICS) Spettrometria di massa Maldi Tof. Mass spectrometry with Mardi Tof

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MODELLI VEGETALI (MODEL PLANT SYSTEMS) Sistema laser per micro dissezione. Laser microdissection of animal and plant tisues. PATOLOGIA ANIMALE Patologia degli animali da laboratorio. Animal Pathology Laboratory

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IMAGING TITOLO (TITLE): MICROSCOPIA A FLUORESCENZA E CONFOCALE (CONFOCAL MICROSCOPE) DURATA (DURATION) : 16 ore (8 teoria – 8 pratica) (16 hrs – 8 theory – 8 practice) CONTENUTI (CONTENT): La microscopia ottica a luce trasmessa e in fluorescenza Markers fluorescenti e preparazione del campione Il microscopio confocale (basi teoriche) Le immagini digitali (introduzione) Colocalizzazione e Fluorescence Resonance Energy Transfer Acquisizioni lambda e separazione spettrale Fluorescence Recovery After Photobleaching

transmitted light fluorescence microscopy Fluorescent markers and sample preparation Theoretical basis of confocal microscopy Introduction to Digital Imaging Colocalization and Fluorescence Resonance Energy Transfer lambda Acquisitions and spectral separation Fluorescence Recovery After Photobleaching

TITOLO (TITLE): TECNICHE DI PREPARAZIONE DI CAMPIONI PER LE MICROSCOPIE ELETTRONICHE (ELECTRON MICROSCOPY SAMPLE PREPARATION) DURATA (DURATION): 24 ore (12 teoria – 12 pratica) (24 hrs – 12 theory – 12 practice) CONTENUTI (CONTENT): Preparazione di campioni biologici per la microscopia elettronica a trasmissione Preparazione di campioni non biologici per la microscopia elettronica a trasmissione Ultramicrotomia a temperatura ambiente e a freddo Preparazione di campioni biologici per la microscopia elettronica a scansione: Fissazione, Critical Point Drying, Metallizazione Preparazione materiali non biologici per la microscopia elettronica a scansione

Preparation and imaging of biological samples in the transmission electron microscope Preparation of non-biological samples in the transmission electron microscope Ultra microtome at room temperature and cold room Preparation of biological samples for scanning electron microscopy: fixation, Critical point drying, metallization Preparation of non biological samples for scanning electron microscopy

CONTATTI(CONTACT) : Fondazione Filarete (services@fondazionefilarete.com)

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MICRO e NANO TECNOLOGIE

TITOLO(TITLE) : MICROSCOPIA ELETTRONICA (ELETRON MICROSCOPY) DURATA (DURATION) : 24 ore (12 teoria – 12 pratica) (24 hrs - 12 theory – 12 practice) CONTENUTI (CONTENT): Il corso ha lo scopo di presentare la microscopia elettronica e di poter verificare le potenzialità di tale tecnologia con applicazioni pratiche su campioni di materiali. (The course aims to present the electron microscopy and to verify the potential of this technology with practical applications on material samples) Il microscopio elettronico a trasmissione (TEM): Principi, modalita’ di funzionamento e applicazioni Il microscopio elettronico a scansione (SEM): Principi, modalita’ di funzionamento e applicazioni Il sistema a trasmissione/scansione (STEM): Principi, modalita’ di funzionamento e applicazioni L’analisi elementale in microscopia elettronica a scansione La litografia a fascio elettronico

transmission electron microscope (TEM): principles, mode of operations and applications scanning electron microscope (SEM): principles, mode of operations and applications transmission/scanning system (STEM): principles, mode of operations and applications Scanning Electron Microscopy Examination and Elemental Analysis the electron beam lithography

CONTATTI (CONTACT): Fondazione Filarete (services@fondazionefilarete.com)

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Staminali (STEM CELL) TITOLO(TITLE): TRAINING IN COLTURE CELLULARI LIVELLO BASE: COLTURE DI LINEE CELLULARI E COLTURE PRIMARIE (BASIC LEVEL TRAINING IN CELL CULTURE: CELL LINES AND PRIMARY CULTURES) DURATA (DURATION): Durata 12 ore teorico-pratico (12 hrs theory-practice) CONTENUTI (CONTENT): Lo scopo del corso è quello di illustrare i principi e i metodi che stanno alla base della tecnica e che sono necessari per affrontare qualsivoglia tipo di coltura cellulare, indipendentemente dall’utilizzo che si persegue. Principi base per lavorare in sterilità: attrezzature, manualità, prevenzione delle contaminazioni . Terreni di coltura: composizione, preparazione, filtrazione. Tecniche di microscopia, conta delle cellule e caratterizzazione fenotipica/morfologica. Allestimento di colture primarie: isolamento di campioni cellulari da tessuti normali (ad esempio biopsie muscolari, cutanee), tumorali e sangue. Realizzazione di colture cellulari: in sospensione, monostrato, mezzi semisolidi. Mantenimento di colture cellulari: criogenia, scongelamento e subcoltura. Valutazione della proliferazione cellulare. Valutazione della citotossicità

(The purpose of this course is to illustrate the principles and methods that are necessary to address any type of cell culture. Bacics of sterile working: equipments, environment, protection and prevention of contamination . Colture media: composition, preparation and filtration. Microscopy techniques, cell counts and phenotypic and morphological characterization. Preparation of primary cultures: isolation of cell samples from normal tissues (eg muscle biopsy, skin), tumor and blood. Construction of cell cultures: suspension, monolayer, semi-solid media. Maintenance of cell cultures: cryogenics, thawing and subculturing. Assessment of cell proliferation. Evaluation of cytotoxicity)

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TITOLO (TITLE): TRAINING IN COLTURE CELLULARI LIVELLO AVANZATO: ISOLAMENTO E COLTURA DI CELLULE STAMINALI SOMATICHE (ADVANCED TRAINING IN CELL CULTURE: CULTURE AND ISOLATION OF SOMATIC STEM CELLS) DURATA (DURATION): 16 ore (16 hrs) CONTENUTI (CONTENT): Il corso si propone di impartire approfondite conoscenze delle caratteristiche biologiche intrinseche e delle potenzialità di manipolazione anche a scopo terapeutico delle cellule staminali somatiche. Tecniche e strumenti per la preparazione del campione cellulare. Trattamento dei tessuti murini ed umani (muscolo, cute, cervello, sangue) da cui isolare cellule staminali somatiche. Sorting immuno-magnetico per l’isolamento di particolari popolazioni di cellule staminali. Tecniche di microscopia, conta delle cellule e caratterizzazione fenotipica morfologica. Terreni di coltura: composizione, preparazione, filtrazione specifici per cellule staminali. Le basi teoriche e pratiche della preparazione, mantenimento e differenziamento di cellule staminali adulte. La crioconservazione di cellule staminali. Valutazione della vitalità e della proliferazione delle cellule staminali isolate. Valutazione della clonogenicità delle cellule staminali. (The course aims to impart detailed knowledge of the intrinsic biological characteristics and potential for therapeutic manipulation also of somatic stem cells. Techniques and tools for the preparation of the sample cell. Treatment of murine and human tissues (muscle, skin, brain, blood) from which to isolate somatic stem cells. Immuno-magnetic sorting for isolation of specific stem cell populations. Microscopy techniques, cell counts and morphological phenotypic characterization. Cell Media: composition, preparation, filtration for stem cells. The theoretical and practical bases for the preparation, maintenance and differentiation of adult stem cells. The cryopreservation of stem cells. Assessment of viability and the proliferation of isolated stem cells. Assessment of clonogenicity of stem cells.

CONTATTI (CONTACT): Fondazione Filarete (services@fondazionefilarete.com)

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Modelli animali (ANIMAL MODEL)

TITOLO (TITLE): IMAGING MOLECOLARE IN DRUG DISCOVERY E SVILUPPO PRE CLINICO (MOLECULAR IMAGING IN DRUG DISCOVERY AND PRE-CLINICAL DEVELOPMENT) DURATA (DURATION): 16 ore (8 teoria – 8 pratica) (16 hrs – 8 theory 8 practice) CONTENUTI (CONTENT): L’imaging funzionale e molecolare nelle indagini biologiche, nella farmacologia e nella medicina. Gli strumenti e il supporto informatico necessari. Il drug discovery in oncologia e sistema nervoso centrale. Meccanismi predittivi di tossicità ed efficacia del farmaco nella sviluppo preclinico. Sezioni teoriche e pratiche. (Functional imaging investigations and molecular biology, pharmacology and medicine. Tools and IT support required. The drug discovery in oncology and central nervous system. Predictive mechanisms of toxicity and efficacy in preclinical development. Theoretical and practical sections.)

CONTATTI (CONTACT): Fondazione Filarete (services@fondazionefilarete.com)

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Modelli Cellulari (CNS CELL MODES)

TITOLO (TITLE): CULTURE PRIMARIE DEL SISTEMA NERVOSO CENTRALE: ALLESTIMENTO E METODI DI STUDIO (PRIMARY CNS CELL CULTURE: STUDY DESIGN AND METHODS) DURATA (DURATION): Durata 12 ore teorico-pratico (12 hrs theory&practice) CONTENUTI (CONTENT): Lo scopo del corso è quello di illustrare i principi alla base della preparazione di colture primarie del Sistema Nervoso Centrale e le tecniche per la loro caratterizzazione funzionale. Saranno descritti i vari tipi di cellule del Sistema Nervoso Centrale (neuroni, astrociti, microglia) e i metodi per il loro isolamento, i vari tipi di substrato di adesione e terreni di coltura, le tecniche di dissezione. Saranno inoltre descritti i principali metodi per la caratterizzazione morfologica e funzionale delle colture: tecniche di immunocitochimica, metodi per lo studio del riciclo delle vescicole sinaptiche, tecniche elettrofisiologiche e di imaging per il calcio. Saranno descritte le principali caratteristiche delle colture pure e delle colture miste.

(The purpose of this course is to illustrate the preparation principles of primary cultures of central nervous system and techniques for their functional characterization. We will describe the various types of CNS cells (neurons, astrocytes, microglia) and methods for their isolation, the different types of substrate adhesion and growth media, the techniques of dissection. We will also describe the main methods for morphological and functional cultures: immunocytochemistry techniques, methods for studying the recycling of synaptic vesicles, calcium electrophysiological and imaging techniques. We will describe the main characteristics of pure cultures and mixed cultures.)

CONTATTI (CONTACT): Fondazione Filarete (services@fondazionefilarete.com)

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TITOLO (TITLE): CULTURE DI LINEE CELLULATI E METODI DI TRASFEZIONE(CELL LINES CULTURE AND METHODS OF TRANSFECTION) DURATA (DURATION): Durata 8 ore teorico-pratico (8hrs theory&practice) CONTENUTI (CONTENT): Lo scopo del corso è quello di illustrare i metodi utilizzati per il mantenimento di linee cellulari e per la loro trasfezione. Saranno descritti i principi base e le attrezzature necessarie per lavorare in sterilità e prevenire le contaminazioni, i tipi di terreni di coltura e i substrati di adesione, le tecniche di passaggio e di conta, il congelamento e lo scongelamento. Saranno inoltre illustrati i metodi di trasfezione transiente (calcio-fosfato, elettroporazione, lipidi cationici) e le tecniche per l’ottenimento di linee cellulari trasfettate stabilmente. Saranno infine descritte le tecniche microscopiche per l’analisi morfologica delle linee cellulari. (The aim of the course is to illustrate the methods used for the maintenance of cell lines and their transfection. We will describe the basic principles and equipment needed to work in sterility and prevention of contamination, types of culture media and substrate adhesion, techniques and step counts, freezing and thawing. We will also visit the methods of transient transfection (calcium phosphate, electroporation, cationic lipids) and techniques for obtaining cell lines stably transfected. We will finally describe techniques for microscopic morphological analysis of cell lines))

CONTATTI (CONTACT): Fondazione Filarete (services@fondazionefilarete.com)

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PROTEOMICA (PROTEOMICS)

TITOLO (TITLE): CORSO TEORICO PRATICO DI SPETTROMETRIA DI MASSA MALDI TOF APPLICATA ALLA PROTEOMICA: LIVELLO BASE (TRAINING COURSE ON MALDI TOF MASS SPECTROMETRY: BASIC LEVEL) DURATA (DURATION): 8 ore teorico – pratiche ( 8hrs theory and practice) CONTENUTI (CONTENT): Introduzione alla spettrometria di massa MALDI-TOF (basi teoriche) Preparazione del campione Acquisizione in modalità lineare e reflectron Identificazione di proteine mediante fingerprint di massa e utilizzo di banche dati proteiche Studi di modificazioni post-traduzionali mediante spettrometria di massa MALDI-TOF

• • • • •

Introduction to MALDI-TOF Sample preparation Acquisition in linear mode and reflectron Identification of proteins by mass fingerprint and use of protein databases Studies of post-translational modifications by mass spectrometry MALDI-TOF

CONTATTI (CONTACT): Fondazione Filarete (services@fondazionefilarete.com)

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modelli vegetali (PLANT MODELS)

TITOLO (TITLE) : CORSO TEORICO PRATICO DI SISTEMA LASER PER MICRODISSEZIONE (THEORETICAL AND PRACTICAL COURSE ON LASER SYSTEM MICRODISSECTION) DURATA (DURATION): 10 ore teorico – pratiche (10 hrs theory&practice) CONTENUTI (CONTENT): Introduzione alla tecnologia di micro dissezione Preparazione del campione Estrazione Dna/Rna dai tessuti/cellule dissecati

Introducion on micro dissection technology Sample preparation Extraction of DNA/RNA from tissue/cells dissected

CONTATTI (CONTACT): Fondazione Filarete (services@fondazionefilarete.com)

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PATOLOGIA ANIMALE (ANIMAL PATHOLOGY)

TITOLO(TITLE): PATOLOGIA DEGLI ANIMALI DA LABORATORIO (ANIMAL PATHOLOGY LAB) DURATA(DURATION): 8 ore (4 teoria – 4 pratica) (8hrs – 4 theory – 4 practice) CONTENUTI (CONTENT): L’attività didattica proposta ha lo scopo di fornire le basi metodologiche ed interpretative sulle quali si fonda l’analisi patologica dei roditori comunemente utilizzati nella ricerca biomedica. Tecniche necroscopiche: interpretazione dei reperti macroscopici e prelievo di campioni biologici idonei Analisi istopatologica: fissazione dei campioni e modalità di trimming Analisi istopatologica: processazione ed interpretazione dei preparati Analisi istochimica ed immunoistochimica

The training course is intended to provide interpretative and methodological bases on which it relies pathological analysis of rodents commonly used in biomedical research. Necropsy techniques: interpretation of macroscopic findings and biological samples preparation Histopathologic analysis: standard settings and trimming rules Histopathologic analysis: Processing and interpretation of the preparations Analysis histochemistry and immunohistochemistry

CONTATTI (CONTACT): Fondazione Filarete (services@fondazionefilarete.com)

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Sommario / Table of Content INSTRUMENT LIST..................................................................................... 63 IMAGING......................................................................................................................................... 68 SPECTROPHOTOMETRY .................................................................................................................. 82 SEQUENCING .................................................................................................................................. 86 GENOTYPING .................................................................................................................................. 88 REAL TIME PCR ............................................................................................................................... 92 MOLECULAR BIOLOGY IMAGING.................................................................................................... 94 HISTOLOGY ..................................................................................................................................... 98 FLUORESCENCE MICROSCOPY ...................................................................................................... 107 CYTOFLUORIMETER ...................................................................................................................... 110 SINGLE CELL PATCH CLAMP ELECTROPHYSIOLOGY ..................................................................... 111 PROTEOMICS ................................................................................................................................ 113 SURFACE FUNCTIONALIZATION ................................................................................................................. 125 THIN FILMS DEPOSITION ........................................................................................................................... 125 OTHER INSTRUMENTS ............................................................................................................................... 129

CONTACT ....................................................................................................................... 134

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INSTRUMENT LIST DESCRIPTION

SERVICES LINK

IMAGING Scanning Electron Microscopy (SEM) Additional services complete the offer: o Scanning Transmission Electron Microscopy (TEM) o X-Ray Dispersive Analysis for elemental investigation (EDX) (Chemical Microanalysis) o E-Beam Lithography

Micro and Nano Fabbrication

IMAGING Confocal Microscopy

Molecular and Cellular Imaging

IMAGING Micro and Nano Fabbrication Scanning for Microarray IMAGING Micro and Nano Fabbrication Optical Microscopy for material science and device investigation IMAGING Micro and Nano Fabbrication Stylus Profilometry IMAGING Laser Microdissection

Model plant

IMAGING Calcium and sodium imaging setup

CNS Cell model systems

SPECTROPHOTOMETRY Multimode microplate readr with low sample volume

Genomics and BioInformatics

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SPECTROPHOTOMETRY Microplate absorbance reader for 96 well

Animal Models Platform

SPECTROPHOTOMETRY Full-spectum spectrophotometer

Animal Models Platform

SPECTROPHOTOMETRY Scanning Spectrophotometer with narrow bandwidth

All Platforms

SEQUENCING Illumina Genome Analyzer

Genomics and BioInformatics

Illumina Bead Scan Sentrix Array Matrix BeadChip Format

Genomics and BioInformatics

Automatic Liquid handling platmofrms

Genomics and BioInformatics

GENOTYPING

GENOTYPING

REALTIME PCR Fast Real Time PCR with 96-384 well plate compatibility (Micro Fluid Card Format)

Animal Models Platform

MOLECULAR BIOLOGY IMAGING Advanced chemiluminescence detection system

All Platforms

MOLECULAR BIOLOGY IMAGING Imaging system from 1-D and 2-D gels to chemiluminescent blots, microplates and autoradiograms

All Platforms

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MOLECULAR BIOLOGY IMAGING Automated electrophoresis station

Animal Models Platform

MOLECULAR BIOLOGY IMAGING Expandable multimode reader (Luminescence, Fluorimeter)

Animal Models Platform

HISTOLOGY Tissue processor Embedding system Fully automated rotary microtome Optical microscope with digital camera

Animal Pathology Laboratory

FLUORESCENCE MICROSCOPY Upright microscope Manual inverted microscope

Animal Models Platform

FLUORESCENCE MICROSCOPY Automated Inverted Microscope

CNS cell model system

FLUORESCENCE MICROSCOPY Inverted microscope with LED

Stem cells

FLUORESCENCE MICROSCOPY Stereomicroscope

Model plant systems

CYTOFLUORIMETER Cytomics

Stem cells

SINGLE CELL PATCH CLAMP Multiclamp

CNS cell model system

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PROTEOMICS 1D and 2D electrophoresis

Proteomics

Western blotting

Proteomics

Gel Image Analysis

Proteomics

LC-ESI Mass Spectrometry

Proteomics

MALDI TOF TOF Mass Spectrometry

Proteomics

PROTEOMICS

PROTEOMICS

PROTEOMICS

PROTEOMICS

PROTEOMICS Automated proteins/peptides sequencer

Proteomics

PROTEOMICS Amino acid analysis

Proteomics

SURFACE FUNCTIONALIZATION Cleanrooms

Micro and Nano Fabbrication

SURFACE FUNCTIONALIZATION Spin coater

Micro and Nano Fabbrication

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SURFACE FUNCTIONALIZATION Nanostructured thin films deposition system

Micro and Nano Fabbrication

SURFACE FUNCTIONALIZATION Evaporator for thin film deposition

Micro and Nano Fabbrication

SURFACE FUNCTIONALIZATION Plasma system

Micro and Nano Fabbrication

OTHER INSTRUMENTS Bioanalyzer for electrophoresis

Genomics and BioInformatics

OTHER INSTRUMENTS Gas Exchange system

Model plant systems

OTHER INSTRUMENTS Square wave electroporator

CNS cell model system

OTHER INSTRUMENTS Auto Densi-Flow Density Gradient Fractionator

CNS cell model system

OTHER INSTRUMENTS Cytospin

CNS cell model system

Spotter

Micro and Nano Fabbrication

OTHER INSTRUMENTS

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IMAGING Aim of this service is to assist all groups and visitors through the microscopic imaging process: project planning, sample preparation, staining, microscope selection and use, image processing, presentation, data transfer and storage. To fulfill this purpose we propose different kind of imaging approaches:

Scanning Electron Microscopy Confocal Microscopy Standard Optical Microscopy Microarray Imaging Stylus Profilometry Laser Microdissection Calcium and sodium imaging setup

Additional services related to the Scanning Electron Microscopy facility complete the offer:

Scanning Transmission Electron Microscopy X-Ray Dispersive Analysis for elemental investigation E-Beam Lithography

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Scanning Electron Microscopy Technical Specifications: ZEISS SIGMA (May 2009) Thermal Field Emission Acceleration voltage: 0,1 – 30 kV Resolution: 1.5nm@20kV - 1.7nm@15kV - 3.0nm@1kV Current: 4pA-40nA Magnification: 12x – 500000x Sample dimensions up to 125mm x 125mm

Laboratory equipment Electron beam evaporator for samples metallization. Under-vacuum stocking system for the samples. Fields of application: Material Science - Every kind of material, even insulating ones, can be visualized with very high spatial resolution. Heterostructures and complex morphologies can be easily undisclosed. Polymers, glass fibers and plastics can be observed without the need for a metallization process MEMS Imaging - Micro and nanofabricated devices can be easily inspectioned. The high resolution of this instrument is helpful in characterizing the cleanness of a machining process down to a few nanometers features. Very low currents can be used to obtain images with low noise level in case of materials sensitive to electron exposure. Biomedicine - This microscope provides surface views of whole structure of specimen. In the biological sciences, the applications of SEM have enormously increased in last few years and this is mainly due to the fact that the preparation of specimen is fairly rapid and simple and the system can give images with an appreciable contrast. Whole small organisms, animal and plant tissues as well as cells cultured on a variety of substrates can be analyzed with the Zeiss Sigma SEM even without the need for a metallization process.

Local Contacts: Gero Bongiorno (gero.bongiorno@fondazionefilarete.com) Davide Marchesi (davide.marchesi@fondazionefilarete.com) Maura Francolini (maura.francolini@fondazionefilarete.com)

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Texture of a glass fiber filter

Fly eye

Monolayer of selfpacked monodispersed 500nm polystyrene beads

Neuronal cell

Drosophila

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Scanning Transmission Electron Microscopy Technical Specifications: Zeiss Multi-mode STEM Detection System. With its design it allows the introduction in the SEM chamber of up to 12 TEM grids at the same time. This detector can be used in combination with the EDX analysis system. Laboratory equipment:

Excitatory synapses in cultured hippocampal neurons

A cell culture laboratory, a wet laboratory, a laboratory fully equipped for ultramicrotomy and an histology facility are available for samples preparation. Fields of applications: Biomedicine Ultrastructural analyses of tissues and cells of animal and plant origin; immunolocalization of proteins and nucleic acids at the ultrastructural level; correlative microscopy. Material Sciences Investigation of multilayered structures, nanostructured materials and composite materials like polymers charged with nanoparticles.

Electron Tomography of an Endoplasmic Reticulum Exit Site (S. Lanzavecchia & M. Francolini)

Local Contacts: Maura Francolini (maura.francolini@fondazionefilarete.com)

Rat Soleus neuromuscular junction

Gero Bongiorno (gero.bongiorno@fondazionefilarete.com) Davide Marchesi (davide.marchesi@fondazionefilarete.com)

TEM micrograph of Pd nanoparticles deposited and included in PMMA

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Scanning Electron Microscopy with Chemical Microanalysis. Technical Specifications: Bruker Quantax 400 with a 30mm2 XFlash silicon drift detector Working in combination with the Sigma Zeiss SEM in both working modes (SEM and STEM modes) is a modular and universal EDX system for qualitative and quantitative microanalysis. The system’s standardless quantification software enables manual, automatic or interactive spectra evaluation and provides reliable results for specimens with polished or irregular surfaces, thin layers and particles. An advantage of the system is the XFlash detector that provides high throughput rates and improved light element detection capabilities with respect to older EDX systems. An image processing utility as well as a fully quantitative line scan and mapping (with submicrometric resolution) with hyper spectral database are a substantial part of the instrument. The system is able to produce maps of chemical contrast with submicrometric spatial resolution. A flexible project management package and various options for quick report generation are present.

+

=

EDX analysis of an antistatic hydraulic filter composed of both glass microfibers and iron-based conductive fibers Local Contacts: Gero Bongiorno (gero.bongiorno@fondazionefilarete.com) Davide Marchesi (davide.marchesi@fondazionefilarete.com)

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Electron Beam Lithography

Technical Specifications:

The Raith Elphy Quantum pattern generator is interfaced with the Scanning Electron Microscope and allows the machining of surfaces with submicrometric resolutions through classic lithographic approaches, lift-off procedures, etc. Machining patterns can be designed with the relative CAD software. Profiles provided in jpeg electronic format can also be handled and impressed on surfaces. Suitable for the realization of devices integrating electric circuits, microfluidic elements and sensing elements. Can be used on many different kinds of materials including silicon, glass, polymers, hydrogels, etc.

Sequence of different auto-centered e-beam exposures for the production of an array of transistors

Patterns produced in polymers for cell culturing and microfluidic devices.

Local Contacts: Gero Bongiorno (gero.bongiorno@fondazionefilarete.com) Davide Marchesi (davide.marchesi@fondazionefilarete.com)

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Optical Microscopy for Materials Science and Device Investigation

Technical Specifications:

Leica DM6000 M with Fluorescence Imaging Setup This optical microscope is specifically designed for industrial applications. It works with reflected light and operates in bright field mode, dark field mode and fluorescence mode. The instrument is fully motorized (x-y-z movements included) and controlled by software or by commands integrated in the microscope itself. A high-speed digital camera allows to collect images on a pc and to control the instrument remotely.

Local Contacts: Gero Bongiorno (gero.bongiorno@fondazionefilarete.com)

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Stylus Profilometry Technical Specifications: Veeco Dektak150 Surface Profiler with low-force head and Advanced Automation setup. This instrument accurately characterizes film thickness, roughness, stress and defects on samples up to 150 mm thick. The system features long-scanning capability and industry-leading data analysis software. The system is equipped with a package that allows the utilization of forces less than 1.0mg (full range 15mg - 0.03mg) suitable for working with soft samples. Z range up to 1mm is suitable in case of measuring the thickness of thick samples or imaging films with very high roughness. The X-Y (150mm x 150mm) automated stage delivers programmability of over 200 locations and 3D mapping capability.

Analysis of a polymeric scratched surface and of a 10cents of Euro coin

Local Contacts: Gero Bongiorno (gero.bongiorno@fondazionefilarete.com)

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Scanner for Microarrays Technical Specifications: Tecan PowerScanner - 4Âľm resolution - 48 slides autoloader - dual channel 635-532nm This instrument allows to scan high density microarrays and is compatible with many different systems available on the market. The PowerScanner is able to operate on both transparent and opaque slides (like for example nitrocellulose) With a double laser light source (635 and 532 nm) and a double PMT detector the system is able to scan the slides in parallel at both wavelenghts. The 48 slides autoloader and the autogain adjusting software allow the full automatization of the acquisition in high-throughput regime.

Local Contacts: Gero Bongiorno (gero.bongiorno@fondazionefilarete.com)

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Confocal Microscopy Leica TCS SPE UV confocal microscope is designed for fixed sample multicolor imaging, colocalization studies, xyz acquisitions and 3D rendering; it is equipped with an upright microscope with 10X, 40X and 63X ACS objectives that are optimally corrected for chromatic aberrations; solid state lasers (405, 488, 532 and 633 nm) and one spectral confocal channel.

Local Contacts: Simona Rodighiero (simona.rodighiero@fondazionefilarete.com)

Maura Francolini (maura.francolini@fondazionefilarete.com)

Leica TCS SP5 AOBS confocal microscope is suitable for fixed samples but is specifically designed for live imaging due to the presence of a fast scanning module (resonant scanner); it is equipped with an inverted microscope (10X, 20X, 40X PlanApo, 63X PlanApo) with gas and solid state lasers (excitation range from 405 to 633nm); 3 spectral confocal channels; conventional and resonant scanner (25 frames per sec at 512x512); microscope climatic chamber to perform time-lapse experiments in environmental controlled conditions and perfusion system. To overcome the limitations of resolution in optical microscopy imposed by the diffraction of light, we employ technologies that detect the transfer of energy from donor to acceptor fluorophores thus allowing the determination of molecule interactions- Fluorescence Resonance Energy Transfer (FRET)-, while Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) provide strong information about mobility and/or diffusion rates of molecole and compounds in living systems. We routinely perform functional tests for anion transporters and channels.

Confocal microscopy images of PC12 cells grown on EBL patterned network of microwells colligated by microchannels

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Acetylcholine Receptors of Rat Soleus neuromuscular junction labelled with TRITC-Bungarotoxin

Biocytin-labelled murine pyramidal neuron. Scale bar 20 um.

in tobacco pollen tubes. Scale bar 10 um.

Acceptor photobleaching experiments in HEK cells expressing the tandem protein ECFP-EYFP.

Excitatory (red) and inhibitory (green and blue) synapses in hyppocampal neurons in colture. Scale bar 10 um.

Pulse-chase experiments using a membrane selective dye

Iodide transport experiments in HEK cells expressing SLC26A4 (pendrin) and EYFP as intracellular iodide sensor.

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Calcium and Sodium Imaging

Overview Calcium and sodium imaging is a technique which typically combines microscopy with the use of ion sensitive fluorescent dyes in order to measure and visualize intracellular ion concentrations. Ion sensitive dyes are fluorescent molecules which reversibly bind to specific ions. These dyes are very sensitive to any change in ion concentration. Depending on the experimental requirements, different detector systems can be used to obtain high resolution information on the time course and/or on the spatial localization of events (high temporal resolution and high spatial resolution). Laboratory equipment The core of the Calcium/Sodium imaging facility is the Imaging Workbench 6 (IW6) that is a proven program for multichannel dynamic fluorescence image acquisition and analysis, with precise control of wavelength switchers (Polychrome V fast monochromator ) and of other external equipments during acquisition. IW6 also allows a flexible review and data extraction during analysis. The software package is set-up on an upright Leica DMI6000 B microscope equipped with ultrafast, highly sensitive Andor iXonEM CCD Camera and the following objectives: 10X, 63X, and a 40X objective APO Lambda BLU that is specifically designed for Fura imaging. Technical Features: Controls z-axis drives for focus and position adjustment during experiments Fully supports Andor iXon camera, at highest speeds and with all features, e.g. iCam for fastest exposure changes Supports OptoSplit image splitter from Cairn Research Support for OptoLED solid state illumination system Easily select which ROI traces you want to show/hide in the Graph window Equipped with high precision syringe pumps and valves for monitoring real time microfluidic experiments (neMESYS). Applications: Ion imaging High temporal resolution experiments Long exposure thanks to high sensitivity FRET Multiple dyes Ratiometric and nonratiometric Video rate Time lapse Microfluidic assays on living cells

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Pseudocolor images showing calcium changes in neurons (circles) and astrocytes (triangle). Red arrows indicate direction of calcium waves along astrocytes in astrocyte-neuron cocultures.

Temporal analysis of synchronous calcium oscillations recorded from the soma of two distinct neurons present in the field (left panel). Kinetics of calcium changes in two astrocytes in the field (right panel).

Local Contacts: Elisabetta Menna (elisabetta.menna@fondazionefilarete.com) Michela Matteoli (michela.matteoli@fondazionefilarete)

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Laser Microdissection

Scanning Stage repositioning

precision

of

2

µm

Auto focus. Serial section cutting Laser movement by optics not by mechanics Specially developed, wide range of objectives and automated CCIC (1.25x – 150x) Integrated solution for everybody’s needs Automated Fluorescence Non-contact stress-free preparation Transport by gravity (upright microscope) Overview The new, state-of-the-art laser Microdissection system from Leica Microsystems satisfies the requirements of even the most modern research labs. Highest precision, High-speed, integrated solution for everybody’s needs and intelligent automation characterize the Microdissection system Leica LMD6000 laser microdissection microscope, with innovations in all areas required for collecting material for analysis.

No mechanical or physical forces are needed Live cell-cutting module Local Contacts: Massimo Galbiati (massimo.galbiati@fondazionefilarete.com) Domenico Allegra (domenico.allegra@fondazionefilarete.com)

Key Features High-speed Microdissection - The fastest way from tissue section to reaction buffer. New Diode laser for highest cutting speed, power and precision Automated multi well positioning and well inspection function. Multi slide holder for higher productivity Automated cell detection and collection SW Cutting over all magnifications in one step High precision systems – When every µm counts

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SPECTROPHOTOMETRY Tecan CM Infinite® M 200 NanoQuant

The Infinite 200 NanoQuant is a multimode microplate reader developed specifically for absorbance applications with low sample volume This sensitive instrument is equipped with UVstable filters, and can detect DNA concentrations as low as 1 ng/μl. The Infinite 200 NanoQuant can be used for a broad range of applications as for example measuring DNA- or RNAquantification or quality control and labeling efficiency. It delivers excellent sensitivity, multiplexing capability and high format flexibility including 6 – 384-well microplates as well as halfarea plates. In the NanoQuant plate a special quartz optic for each of the 16 samples guarantees outstanding performance and a high rate of reproducibility. The plate offers quick and easy cleaning to reduce the risk of cross contamination. In addition after a working day the plate can be even cleaned in an ultra sonic water bath for additional efficient cleaning. The NanoQuant plate is compatible with an eight-channel pipette to easy dispense the samples on the 16 positions of the tool.

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Tecan CM Sunrise™

Sunrise is a versatile microplate absorbance reader for 96-well plates providing all the functionality needed for the numerous applications in diagnostics, biotechnology and research laboratories · · · · · · · · · · ·

Supports ELISA- & kinetics-, as well as agglutination reading Selection of user interface: Netbook* or Touchscreen Selection of eight languages for easy operation 3 year warranty as Tecan´s commitment to quality Reads plate in only 6 sec. via advanced 12-channel optics Powered by smart MagellanTM data analysis software Quick access to your program favourite via start-screen Advanced curve-fitting & plate-to-plate QC functions IQ / OQ procedure & documentation Designed to meet IVDD 98/79/EC & FDA´s 21 CFR part 11** Filter 405, 450, 492 and 620 nm

A set of options such as temperature control up to 42°C, a gradient filter for wavelengths scanning and a barcode scanner allow to customize the Sunrise to your application needs. The compact space saving design makes the Sunrise ideal for stand-alone use, as well as integration into automated ELISA-systems such as the Freedom EVOlyzer®.

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Nanodrop ND-1000

The NanoDrop ND-1000 is a full-spectrum spectrophotometer (UV and visible spectrum, 220-750 nm) for measuring the absorbance of DNA, RNA, proteins and dyes. The newly developed sample retention system allows for measurement of small samples volumes: the sample droplet is held in place by surface tension when it is slightly compressed between the pedestal and the sample arm; this generates the defined pathway of 1 mm. The spectrum measurement is then performed with two optical fibers installed in the pedestal (emitting light of a Xenon lamp) and the sample arm (spectrometer with linear CCD array). Quantification is performed based on the spectrum measurement at the defined pathway of 1 mm. DNA, RNA, protein or dye in 1 µl can be measured without using a cuvette or capillaries. The NanoDrop ND-1000 is a small apparatus (20 cm x 14 cm). A measurement only takes 10 sec. Sample measurements are possible over a wide range of concentrations, e.g. double-stranded (ds) DNA: 2-3700 ng/µl, single-stranded (ss) DNA: ≤ 2400 ng/µl, RNA: ≤3000 ng/µl, and protein: 0.10-100 mg/ml BSA (at 280 nm); this is 50x higher than ranges of normally used spectrophotometers. Once the measurement is complete,the surfaces are simply wiped witha lint-free lab wipe.

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Beckman DU速 730 UV/Vis Scanning Spectrophotometer

The narrow bandwidth allows you to scan your samples with excellent resolution. With its focused-beam design, the system provides optimal and reproducible results for small samples

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SEQUENCING Illumina Genome Analyzer IIx

Genome Analyer II x

Illumina sequencing technology leverages clonal array formation and proprietary reversible terminator technology for rapid and accurate large-scale sequencing. Illumina sequencing technology has the ability to generate several gigabases of DNA sequence per run, providing researchers with the opportunity to sequence even large mammalian genomes in weeks rather than years. Leveraging this technology, researchers can potentially resequence genomes for less than 1% of their current costs. With the capacity to accommodate many samples per flow cell (actually 96), runs can be tailored to the demands of a wide range of applications. The innovative and flexible sequencing system offers a simple and fast workflow and enables a broad array of applications in genomics, transcriptomics, and epigenomics as follow: · · · · · ·

Target Sequencing; Resequencing De-novo sequencing RNA and miRNA sequencing Methylation profile ChipSeq

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Cluster generation Sequencing templates are immobilized on a proprietary flow cell surface designed to present the DNA in a manner that facilitates access to enzymes while ensuring high stability of surface-bound template and low non-specific binding of fluorescently labeled nucleotides. Solid-phase amplification creates up to 1,000 identical copies of each single template molecule in close proximity (diameter of one micron or less). Because this process does not involve photolithography, mechanical spotting, or positioning of beads into wells, densities on the order of ten million single-molecule clusters per square centimeter are achieved.

Left: cluster generation. Right: Flow cell

Sequencing by synthesis Illumina sequencing uses four proprietary fluorescently-labeled modified nucleotides to sequence the tens of millions of clusters present on the flow cell surface. These nucleotides, specially designed with a reversible termination property, allow each cycle of the sequencing reaction to occur simultaneously for all clusters in the presence of all four nucleotides (A, C, T, G). In each cycle, the polymerase is able to select the correct base to incorporate, with the Natural competition between all four alternatives leading to higher accuracy than methods where only one nucleotide is present in the reaction mix at a time. Sequences where a particular base is repeated (e.g., homopolymers) are addressed like any other sequence and resolved with high accuracy.

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GENOTYPING Illumina Bead Scan

Illuminaâ&#x20AC;&#x2122;s BeadArray Reader is a confocal scanner with < 1 micron resolution for use with Sentrix Array Matrices and Sentrix BeadChips. The BeadArray Reader offers broad dynamic range, high sensitivity, and a low limit of detection: these metrics are particularly important for users planning to study gene expression and other applications that are supported by BeadArray technology and that demand high scanner performance. The Illumina BeadArray Technology supports Gene Expression profiling of focused sets of genes and whole genomes and SNP Genotyping, enabling a wide range of sample throughput and broad experimental scale. The BeadArray Reader is equipped with a Dual Autoloader for the automation of chip loading on the BeadStation. Illumina BeadArray Technology Illumina's microarray technology is based on arrays of randomly assembled glass (silica) beads. Beads are 3 microns in diameter and randomly assembled on the arrays uniformly spaced about 5.7 microns apart. All the beads have about 1 milion oligonucleotides covalently attached to the surface and all oligos on each bead have the same sequence. Beads with the same oligonucleotide sequence have the same bead type and each bead type is produced in manufacturing lots of several million. Full-length oligonucleotides are coupled to the beads through an amine linkage and different bead types are pooled together to form assay panels for 96 to over a million targets. BeadArray technology comes in 2 formats: the Sentrix Array Matrix and the Bead Chip.

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Sentrix Array matrix The 96-well Sentrix Array Matrix consists of bundles of fiber optic cables. The Sentrix Array Matrix is the size of a standard 96-well plate in which each well in the Sentrix Array Matrix is a bundle of optical fiber cables. One end of the cables detects light, the other end has a well etched into it, where a bead can fit. Each array has about 50,000 beads coming from a bead pool consisting of a mixture of about 1520 different bead types: each bead type is present about 30 times, 30-fold redundancy.

BeadChip format The BeadChip format consists of a planar silica substrate in a slide format. The silica is coated with a photo-resistant substance and a plasma etching is used to produce uniform wells in the silica surface. Beads are then randomly assembled and held in the wells by Van der Waals forces and by hydrostatic interactions with the walls of the well.

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Sentrix Array Matrix and BeadChip formats

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Tecan Freedom EVO 75 liquid handler

The Freedom EVO 75 open liquid handling platform has been developed to handle repetitive liquid transfer tasks. This small footprint robotic workstation has huge flexibility and thanks to its freely configurable system, can support many applications such as: · · · · · · ·

Nucleic acid extraction ELISA assay Setting up PCR and sequencing reactions Serial dilutions MALDI spotting Immunostaining protocols and in situ hybridization (ISH) Capillary electrophoresis set-up

The liquid system is a central component of the pipetting function. The uncompressible liquid transmits the precise movement of the diluter pistons to the tip and with the Flow-Thru Technology, pipetting is not only highly precise, but the channels flushed with system liquid remain clean at all times. By sharing the software as well as the major mechanical and electronic components, Freedom EVO 75 can interact with other Tecan devices for detection, separation, plate handling and storage as well as other third party devices.

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REAL TIME PCR Applied Biosystems 7900 HT Fast Real-Time PCR System

Applied Biosystems 7500 Real-Time PCR System

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Real-time Polymerase Chain Reaction (rtPCR or quantitative PCR - qPCR) is one of the most powerful and sensitive gene analysis techniques available. It is used for a broad range of applications including : • • • • • • • • • •

quantitative gene expression analysis, SNP analysis, pathogen detection, drug target validation and for measuring RNA interference. Microarray Verification Quality Control and Assay Validation Copy Number Variation MicroRNA Analysis Viral Quantitation siRNA/RNAi experiments

Adavantages in the use of Real-Time PCR versus traditional PCR include: • • • • •

Increased dynamic range of detection No post-PCR processing Detection is capable down to a 2-fold change Data are collected in the exponential growth phase of PCR The cleaved probe provides a permanent record amplification of an amplicon

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MOLECULAR BIOLOGY IMAGING Biorad Molecular Imager ChemiDoc XRS System

The ChemiDoc XRS+ system is the most advanced chemiluminescence detection system in the BioRad gel documentation line. The system features flat fielding technology for superior uniformity and quantitation, and it has the flexibility to image chemiluminescent, fluorescent, and colorimetric samples. The system includes a supersensitive 16-bit CCD camera, light-tight compact darkroom, slide-out transilluminator with preparative mode for added safety when manually visualizing samples, software-driven motorized lens and illumination sources, and powerful, userfriendly Quantity One 1-D analysis software. Unlimited copies of Quantity One Basic software, available with each system, allow free functionality for sharing, analysis, and presentation of gel data. The ChemiDoc XRS+ system can be used for imaging in a wide variety of applications (western blotting, nucleic acid detection, 2-D gel electrophoresis, dot blotting, densitometry, and colony counting) using a large portfolio of detection methods (Immun-Star HRP and AP chemiluminescence detection as well as ethidium bromide, SYBR Green I, SYPRO Ruby, Cy3, rhodamine, Green Fluorescent Protein, Hoechst, silver, copper, and Coomassie Blue staining).

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Biorad Molecular Imager VersaDoc MP 4000 System

VersaDoc imaging systems are high-quality, flexible instruments that allow imaging of a wide range of samples — from 1-D and 2-D gels to chemiluminescent blots, microplates, and autoradiograms. · A Wide Range of Supported Applications · Proteomic and genomic studies · Protein gels (2-D), including DIGE and other multichannel differential electrophoresis techniques · Nucleic acid and protein gel imaging (1-D) · Multiplex gel and blot imaging · Western, northern, and Southern blot imaging · Quantum dot imaging · Colony counting The standard configuration includes a supersensitive, deeply cooled CCD camera, darkroom, power supply, cables, epi-illumination (blue and white), transillumination (UV), fluorescent reference plate, focusing target, and Quantity One 1-D analysis software. Optional accessories include green and red excitation sources for increased multiplexing capabilities (useful for DIGE and imaging of quantum dots and organic fluroscent secondary antibodies). The VersaDoc MP 4000 system is an ideal imager for proteomic studies, offering maximum flexibility with the ability to image colorimetric and fluorescently stained gels and blots to detect chemiluminescent and other low-light and multiplex or multifluorescent samples. It resolves the finest details of every band or spot. Features include: · 3.2 megapixel CCD with 53 µm resolution · Accurate and quantifiable collection of data with a CV ≤5% · Identification of differences in protein expression using optional PDQuest 2-D analysis software The camera is cooled to absolute temperatures to minimize background noise, enhance the signalto-noise ratio, and maintain reproducible imaging conditions. This high-performance digital camera produces luminescent images of very faint samples (due to either low-abundance samples or faint signal) at speeds faster than exposure times of film-based detection. Features include: · Supercooling to -35°C (absolute) for optimal imaging with low-light applications · True 16-bit data and a dynamic range that covers 5 orders of magnitude to maximize limit of detection · Quantitation of differences in sample abundance using Quantity One 1-D analysis software · Red, green, blue, and UV illumination for multiplex quantitative imaging

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Biorad Experion 700-7002 Automated Electrophoresis Station

The Experion automated electrophoresis station performs all of the steps of gel-based electrophoresis in one compact, durable unit. It automates analysis by combining electrophoresis, staining, destaining, band detection, and imaging into a single, 30 min step. It's an electrophoresis cell, power supply, and imager built into a single device and is designed for easy, precise operation: · · · · · · · ·

Highly accurate laser provides precise fluorescence detection USB port allows easy installation and maximum usability Built-in power supply reduces cost and saves bench space The following types of kits are available for analysis of proteins, RNA, and DNA: Experion starter kits Experion protein analysis kits Experion RNA analysis kits Experion DNA analysis kits

The Experion automated electrophoresis system can be used upstream and downstream of a number of other nucleic acid- and protein-application focused products from Bio-Rad.

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Promega GloMax®-Multi Detection System

The GloMaxR-Multi Detection System is an expandable multimode reader with unbeatable performance. Each detection mode has dedicated optics for the highest versatility without sacrificing performance. The GloMaxR-Multi Detection System can be used as a reader dedicated to a single mode or as a multimode reader. As your application needs expand, the system can easily accept the add-on modules, which offer additional detection modes. This flexibility allows you to customize the system to fit your laboratory needs. Luminescence module · Sensitive to approximately 3 x 10-21 moles of luciferase · 9 log dynamic range · Ideal for cell-based assays · Engineered to minimize sample cross-talk · Direct-to-Excel® data importing: Requires PC to operate · Excel® macros allow simple data analysis for Dual-Luciferase® Assays Fluorimeter module

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HISTOLOGY The Mission The mission of The Mouse & Animal Pathology Lab (MAPLab) is to provide integrated and highly qualified animal pathology service with particular emphasis for the pathobiology of laboratory animal used in biomedical research. In this context, MAPLAb aims at offering its established expertise to support research groups and pharmaceutical companies interested in generation, validation and assessment of animal models recapitulating specific human conditions or used in preclinical efficacy and toxicological studies.

The Staff To pursuit its mission the The MAPLab include a small group of motivated scientists and expert board certified comparative pathologists who are knowledgeable in laboratory animal pathology. name

title

position

Prof. Scanziani Eugenio Dr. Radaelli Enrico Dr. Recordati Camilla Dr. Castiglioni Vittoria Dr. Losa Marco

DVM, Dipl. ECVP DVM, PhD, Dipl. ECVP DVM, PhD, Dipl. ECVP DVM DMedBiotech

Head Pathologist Pathologist Pathologist in training Senior technician

The services of MAPLab include: 1. 2. 3. 4. 5.

Necropsy of laboratory rodents and rabbits Histology on animal tissues; Immunohistochemistry; Image analysis; Other services.

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1. Necropsy of laboratory animals Standardized necropsy and sampling procedures and recognition of gross lesions represent crucial preliminary steps for a successful and reliable interpretation of the entire pathological scenario affecting laboratory animals. Gross lesions can also give important clues to restrict the list of potential differential diagnoses. In some cases gross lesions are so pathognomonic for specific entities that may be sufficient to achieve very accurate diagnostic interpretations 1.1

MAPLab offers:  complete necropsy with organs collection and weight;  immersion fixation of representative specimens from all organs and tissues (perfusion fixation may be also performed for the evaluation of critical tissues such as those of the central nervous system);  digital image documentation of gross lesions, and written reports.

Fig 1.1 – Aged C57BL/6 mouse, lungs; multiple variablesized whitish nodular masses likely representing a spontaneous development of multicentric bronchioloalveolar adenomas/carcinomas.

Fig 1.2 – SCID mouse, thoracic organs; spontaneous thymic lymphoma expanding and infiltrating the cranial mediastinum and compressing lung lobes.

2. Histology on animal tissues Histology is the main technique in pathology. It allows a complete and careful morphological examination of animal tissues for the identification of lesions. Samples are usually submitted as formalin-fixed tissues that are embedded in paraffin, sectioned and stained wit routine (hematoxylin & eosin, fig 1.1) or special stains (Periodic Acid Schiff, Alcian PAS, Toluidine blu, Warthin Starry, Gram, etc). Histological slides are then examined under a light microscope and interpreted by the veterinary pathologist.

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2.1

Laboratory equipment       

2.2

Fully enclosed tissue processor for paraffin embedding Embedding centre Automated rotary microtome Staining centre Optical microscopes Wet tissues archives Paraffin block and slides archives

Fields of application       

histology and pathology on animal tissues phenotyping of Genetically Engineered Mouse (GEM) models setting of animal models used in biomedical research or in preclinical studies identification and quantification of lesions in preclinical efficacy studies identification and quantification of lesions in toxicity studies pathological support during health monitoring programs in animal research facilities identification of "spontaneous" pathological conditions in laboratory animals: due to infective causes (ex. Corynebacterium kutscheri infection in immunocompromised rats): that are strain associated (ex. eosinophilic and granulomatous pneumonia in Brown Norway Rats); that represent incidental findings related to technical procedures (ex. hepatic glicogenosis/lipidosis in non-fasted mice); linked to specific social-environmental factors (ex. male aggression or “pugilistic” male mice).

Fig. 2.1 – Mouse skin; well-differentiated squamous cell carcinoma with formation of characteristic keratin pearls. Hematoxylin-eosin.

Fig. 2.2 – Aged male SD rat treated with d limonene; Accumulation of hyaline droplets consistent with alpha2uglobulin in proximal renal tubules. The formation of alpha2u-globulin-d limonene complex prevents alpha2uglobulin from lysosomal degradation. Hematoxylin-eosin .

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3. Immunohistochemistry Immunohistochemistry (IHC) embodies an essential supportive tool for the characterization of microscopic lesions in addition to standard microscopic approach. IHC is an immunoenzymatic technique that is able to detect antigens in histological sections or cytological samples by using specific primary antibodies.

3.1

Laboratory equipment    

3.2

Refrigerated and frozen bank of primary antibodies Semiautomated staining centre Optical microscopes Slides archives

Fields of application Detection in histological sections of:        

structural proteins (ex. cytokeratins) hormons (ex. prolactin, insulin, calcitonin) receptors (ex. estrogen receptors, progesterone receptor, c-Kit) markers of cell proliferation (ex. Ki67, PCNA) markers of apoptosis (ex. Caspase3) oncogene and tumor suppressor gene products (ex. HIF-1 alpha, p53, β-catenin) markers of leukocyte differentiation (ex. CD3, CD79alpha, CD18) microbial antigens (ex. Helicobacter spp., Leptospira spp.)

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Fig. 3.1 - Cdkn2a null mouse; kidney; hyaline droplet nephropathy; massive reabsorption of lysozyme-positive proteinaceous material by the proximal convoluted renal tubules in a mouse suffering from histiocytic sarcoma. Immunohistochemistry for Lysozyme.

Fig. 3.2 - Cdkn2a null mouse; hepatic histiocytic sarcoma; hepatic cords partially effaced by nodular infiltrates of F4/80-positive neoplastic histiocytes; reactive Kupffer's cells lining the adjacent sinusoidal spaces also express F4/80 antigen. Immunohistochemistry for F4/80.

Fig. 3.3 â&#x20AC;&#x201C; Aged female FVB/N mouse; mammary gland carcinoma; marked loss of beta-catenin immunoreactivity in the anaplastic tumor component. Immunohistochemistry for Beta-catenin.

Fig. 3.4 â&#x20AC;&#x201C; Male DBA/2 mouse; collagen-induced arthritis. synovial membrane moderately expanded by scattered aggregates of reactive synoviocytes intensely positive for MPO. Note also the few MPO-positive neutrophils infiltrating the adjacent tissue. Immunohistochemistry for Myeloperoxidase.

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4. Digital image analysis The achievement of high quality immunohistochemical and histopathological specimens using standardized and reproducible methods allows the application of automated digital image analysis. These innovative computer-based techniques represent objective and reliable systems for the evaluation and quantitation of specific morphologic parameters in labeled tissue sections. Another major advantage of computer-based techniques applied to immunohistochemistry is the avoidance of inter-observer variability in interpreting subtle antigen level changes. With image analysis, microphotographs from histological slides are captured through a digital camera and pictures are analysed using specific softwares to automatically measure parameters such as shape, colour, surface area, number of objects and other parameters. Image analysis is thus employed to exactly quantify lesions or other relevant morphological findings present in histological sections.

4.1

Laboratory equipment  Optical microscope with a digital camera connected to a computer with a specific software.  Electronic archive of digital pictures.

4.2

Fields of application    

Quantification of lesions in efficacy studies Quantification of lesions in toxicity studies Count and measurement of tissue structures and cells. Measurement of histochemical and immunoistochemical positivity.

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Fig. 4.1 - Left: Mouse model of chronic-progressive colitis; DSS-treated C57BL/6 mouse; colonic mucosa; proliferative activity in epithelial cells and infiltrating inflammatory cells is assessed through Ki-67 immunostaining. Right: Application of automated digital image analysis for the calculation of Ki-67-positive (red) versus Ki-67-negative (green) nuclei. Immunohistochemistry for Ki-67.

Fig. 4.2 - Rat model of benign prostate hyperplasia. Left: hematoxylin-eosin staining of rat prostate. Right: Image analysis of prostate epithelial area performed using image analysis.

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5. Other services The following other services offered by the MAPLab in the field of veterinary pathology are available upon request: Evaluation of a single case (the report includes: AFIP style description, morphological diagnosis/es, comment with interpretation of the lesions, references, answers to specific questions) Peer-review of histopathological evaluation Microphotographs of histological lesions In lab practical technical training Preparation or review of scientific papers or internal reports Preparation of oral presentations Organization of courses or scientific meetings Consultation at MAPLab of histoslides of didactic interest with corresponding AFIP style histopathological description, morphological diagnosis/es and comment with interpretation of the lesions

6. Peer rewieved articles published by the scientists of the Mouse and Animal Pathology Laboratory (MAPLab) - year 2009-2010 1. Loss of the actin remodeler Eps8 causes intestinal defects and improved metabolic status in mice. Tocchetti A, Soppo CB, Zani F, Bianchi F, Gagliani MC, Pozzi B, Rozman J, Elvert R, Ehrhardt N, Rathkolb B, Moerth C, Horsch M, Fuchs H, Gailus-Durner V, Beckers J, Klingenspor M, Wolf E, HrabÊ de Angelis M, Scanziani E, Tacchetti C, Scita G, Di Fiore PP, Offenhäuser N. PLoS One. 2010, 5(3):e9468. 2. Suppurative adenitis of preputial glands associated with Corynebacterium mastitidis infection in mice. Radaelli E, Manarolla G, Pisoni G, Balloi A, Aresu L, Sparaciari P, Maggi A, Caniatti M, Scanziani E. J Am Assoc Lab Anim Sci. 2010, 49:69-74. 3. Qualitative evaluation of tortellini meat filling by histology and image analysis. Ghisleni G, Stella S, Radaelli E, Mattiello S, Scanziani E. Int J Food Sci Tech. 2010, 45:265-270. 4. Epithelial-mesenchymal transition in mouse mammary tumorigenesis. Radaelli E, Damonte P, Cardiff RD. Future Oncol. 2009, 5: 1113-27. 5. Cell Lines Derived from Human Parthenogenetic Embryos Can Display Aberrant Centriole Distribution and Altered Expression Levels of Mitotic Spindle Check-point Transcripts. Brevini TA, Pennarossa G, Antonini S, Paffoni A, Tettamanti G, Montemurro T, Radaelli E,

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Lazzari L, Rebulla P, Scanziani E, de Eguileor M, Benvenisty N, Ragni G, Gandolfi F. Stem Cell Rev Rep. 2009, 5:340-352 6. Spatial distribution of Helicobacter spp. in the gastrointestinal tract of dogs. Recordati C, Gualdi V, Craven M, Sala L, Luini M, Lanzoni A, Rishniw M, Simpson KW, Scanziani E. Helicobacter. 2009, 14:180-91. 7. Diagnostic exercise: sudden death in a mouse with experimentally induced acute myeloid leukemia. Radaelli E, Marchesi F, Patton V, Scanziani E. Vet Pathol. 2009, 46:1301-5. 8. Immunohistopathological and neuroimaging characterization of murine orthotopic xenograft models of glioblastoma multiforme recapitulating the most salient features of human disease. Radaelli E, Ceruti R, Patton V, Russo M, Degrassi A, Croci V, Caprera F, Stortini G, Scanziani E, Pesenti E, Alzani R. Histol Histopathol. 2009, 24: 879-91. 9. Mammary tumor phenotypes in wild-type aging female FVB/N mice with pituitary prolactinomas. Radaelli E, Arnold A, Papanikolaou A, Garcia-Fernandez RA, Mattiello S, Scanziani E, Cardiff RD. Vet Pathol. 2009, 46: 736-45. 10. Expression of major histocompatibility complex class II antigens in porcine leptospiral nephritis. Radaelli E, Del Piero F, Aresu L, Sciarrone F, Vicari N, Mattiello S, Tagliabue S, Fabbi M, Scanziani E. Vet Pathol. 2009, 46: 800-9. 11. Expression of class II major histocompatibility complex molecules in chronic pulmonary Mycoplasma bovis infection in cattle. Radaelli E, Luini M, Domeneghini C, Loria GR, Recordati C, Radaelli P, Scanziani E. J Comp Pathol. 2009, 140: 198-202. 12. Role of the molybdoflavoenzyme aldehyde oxidase homolog 2 in the biosynthesis of retinoic acid: generation and characterization of a knockout mouse. Terao M, Kurosaki M, Barzago MM, Fratelli M, Bagnati R, Bastone A, Giudice C, Scanziani E, Mancuso A, Tiveron C, Garattini E. Mol Cell Biol. 2009, 29: 357-77.

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FLUORESCENCE MICROSCOPY Upright Microscope Leica DM2500 •

The Leica DM2500 microscope is the ultimate tool for demanding tasks in pathology, cytology, haematolgy, and even for basic research applications. With its powerful 100W illumination, high-quality optical performance, and state of the art accessories, the Leica DM2500 is especially well suited for challenging goals in pathology or biomedicine that require differential interference contrast or highperformance fluorescence. The bright 100W illumination is particularly beneficial for DIC work. The modular, application-oriented design means that the Leica DM2500 can be configured to fit specific applications and physical requirements of the user

Manual Inverted Microscope Leica DMI3000 B •

The Leica DMI3000 B research microscope supports all transmitted light methods including fluorescence, live cell, time-lapse imaging; highspeed multi-fluorescence optical sectioning, micromanipulation, and more. The viewing angle of the ergonomic tube, a standard feature, can be continuously adjusted to ensure the most comfort for the user. The Leica DMI3000 B’s viewing channel, a notch between the eyepieces, provides a clear view of the specimen regardless of the tube’s position. All of the microscope’s controls are easily accessible for convenient operation.

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Automated Inverted Microscope Leica DMI4000 B The Leica DMI4000 B automated inverted research microscope is ideal for scanning cell and tissue cultures. The system features a fluorescence axis for ultra brilliant fluorescence imaging. •

The internal filter wheel with motorized excitation manager and FIM (Fluorescence Intensity Manager) enables excitation of fluorochromes in less than 20 milliseconds. FIM regulates light intensity at five fixed levels and remembers the setting for each filter cube.

Inverted Microscope with LED Illumination Leica DM IL LED

• •

Samples are focused with the quadruple objective nosepiece. The transmitted light illumination allows the integration of the contrast slide (for phase and modulation contrast) The fluorescence filter cubes perform a matched combination of excitation, reflection, band-pass and barrier filters.

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Stereomicroscope Leica M205 FA

• • •

• • •

Operating comfort and reproducibility through motorization. Intelligent control with SmartTouch™. Encoding for reproducibility and consistency in experiment procedures. Optimal display of your specimens with high-performance objectives. Solid foundation for your research: a stable mechanical construction. Integrated system solutions make life easier

Overview The Leica M205 FA combines the proven TripleBeam® technology with the unique FusionOptics™ concept to open up new dimensions in fluorescence stereomicroscopy. The fully apochromatically optics system, the largest zoom range available on the market, 20.5 : 1, and a resolution to 1050 lp / mm will provide you with a view of details hitherto unknown in stereomicroscopy. The Leica M205 FA allows timeintensive studies of living organisms and documentation of complex image series. Multiple fluorescence images are made possible and instantly reproducible with motorized focus, zoom, filter changer and iris diaphragm.

Key Features • Never before seen: 3D images of highest resolution, brilliance and depth of field. • Largest zoom range in stereomicroscopy. • Brilliant fluorescence images, rich in detail and contrast • Microscopes that grow with your requirements.

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CYTOFLUORIMETER Cytomics FC500 Beckman Coulter

LASERS: · Uniphase Argon ion, 488nm, 20mW output · Coherent Red Solid State Diode, 635nm, 25mW output FLOW CELL: · 150 × 450 micron rectangular channel BioSense · enhanced optics quartz mounted with vertical · (upwards) flow path for superior hydrodynamic · Focusing DETECTORS: · Forward Scatter Detector – Solid state detector with Patented Fourier design providing integrated dual collection angle selection. · Side Scatter Detector – High performance · photodiode · Fluorescent Detectors – High performance · photomultipliers with spectral sensitivity from 185nm-900nm

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SINGLE CELL PATCH CLAMP ELECTROPHYSIOLOGY Overview In the 1950s, scientists began to suspect that single-ion channels existed, but it took them another quarter century to verify it, when in 1974, physicist Erwin Neher and cell physiologist Bert Sakmann invented the "patch clamp" technique , the first device to measure the flow of electrical current through single-ion channels. This technique is based on the possibility to make tight contact with a tiny area, or patch, of neuronal membrane thanks to a glass pipette with a very small opening, which allows the isolation of a patch of membrane electrically from the external solution. Thus, all the ions that flow when a single ion channel opens must flow into the pipette. The resulting electrical current, also very small, can be measured with an ultrasensitive electronic amplifier connected to the pipette. Several recordings configuration of the patch clamp technique enable investigation of macroscopic currents of entire cells as well as elementary single channel currents in microscopic membrane pieces (patches). The important advantage of this method is the possibility to make recording under conditions where voltages, currents and solutions are controlled and can be manipulated during the experiments. So far, patch clamp electrophysiology is the crucial technique that allows to study ion channelsâ&#x20AC;&#x2122; properties and neuronal electrical activity either in resting condition or upon stimulation.

(A) A patch pipette onto pyramidal neurons (B) Representative neuron showing the firing pattern typical of a regular spiking pyramidal neuron recorded in current clamp mode. (C) Gabaergic post-synaptic currents (GPSCs)recorded in a pyramidal neuron, at the indicated Vm

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recorded in voltage clamp configuration. (D) Current-voltage plot of the GPSCs from the same experiment

Laboratory equipment: The core of patch clamp set up is Multiclamp 700B a computer-controlled dual channel resistivefeedback patch clamp and high-speed current clamp amplifier that is controlled by the MultiClamp 700B commander that provides automation of a series of parameters. The Data Acquisition Systems is provided by Digidata 1440, a low-noise digitizer for recording data onto a Windows computer. Digidata 1440A is equipped with pCLAMP Software 10, the most widely used data acquisition and analysis program for the control and recordings of patch clamp experiments. Three separate programs are included: Clampex 10, AxoScope 10, Clampfit 10. The entire set-up is mounted to a manual inverted Leica DM3000B microscope for transmitted light and fluorescence equipped with two Narishige hydraulic micromanipolator WR-6 located onto an antivibration table with Faraday cage that prevents the entry or escape of an electromagnetic field. The fabrication of patch pipettes to make a tight contact with cell are made by the Sutter P-97 Flaming/Brown type pullers and by MF-830 Microforge, by using fire-polishing technology to polish the microelectrode tips with a powerful microscope (525x total magnification) included, for making electrodes with tips less than 2µm. The perfusion system for drug screening is provided by Six Channel Perfusion Valve Control Systems (VC-6; Warner Instruments) Applications: Voltage clamp experiments underlying ionic currents to investigate among others · Vesicular neurotransmitter release process by recording spontaneous electrical activity such as miniature excitatory and inhibitory post-synaptic currents · Neuroprotective or neurotoxic drugs screening · The biophysical properties and the expression of the channel during neuronal development or in some neurodegenerative disease model in vitro Current clamp mode recordings to provide important informations about the channel function in the entire cells for example: · cell excitability by measuring channel activation, inactivation, rest potential and cell spiking pattern and thereshold Double Patch to induce evoked electrical activity and to perform train stimuli protocols to investigate long term potentiation and plasticity mechanisms

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PROTEOMICS The proteomic platform consists of: 

Preparation facility lab for the preparation of samples and separation of complex protein mixtures by electrophoretic (1DE and 2DE) and chromatographic methodologies. Characterization facility lab for mass spectrometric, N-terminal amino acid sequence and amino acid analysis.

R&D activities The main activities of the platform include: 

Characterization of the proteome of cells and tissues including evaluation of protein expression, identification of specific proteins in complex mixtures and characterization of posttranslational modifications;

Analysis of protein-protein interactions and complexes;

Identification of new biomarkers in the clinical and pharmaceutical fields and of antigens for the development of new vaccines by SERPA (serological proteome approach);

Identification of new therapeutic targets for diseases with a high social impact like cancer and neurodegenerative diseases;

Small molecule metabolite profiling of biological samples (metabolomics);

Application in nutritional science: screening for novel functional bioactives, detection and control of food spoilage and allergenic proteins;

Development of new protocols for proteomic analysis based on new nanomaterials;

Quality control of recombinant proteins and synthetic peptides,

Technological services 

Technical advice and services for proteomic analyses including sample preparation, proteins separation by electrophoretic and chromatographic methods, mass spectrometry analyses, western blot analyses, Nterminal sequence analysis, amino acid analysis and qualitative and quantitative differential analysis of samples;

Purification and biochemical characterization of proteins from different sources including evaluation of enzymatic activity.

Renting of equipment for proteomic analyses to customers

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1D and 2D electrophoresis

Technical Specifications: Ettan™ IPGphor™ 3 IEF System from GE Healthcare Run up to 12 IPG strips (7, 11, 13, 18, or 24 cm) simultaneously PROTEAN II xi | XL Multi-Cells from Biorad Run up to 6 home-made or pre-cast large (up to 18.3 x 19.3 cm) gels Mini-PROTEAN® Tetra Cell from Biorad Run up to 4 home-made or pre-cast mini (7 x 8 cm) gels Fields of application:

of growth cycles, comparative examination of physiological and pathological states for classification and diagnosis of disease, monitoring of drug action and other studies involving global qualitative and quantitative evaluation of protein patterns Determination of MW, subunit composition and polymerization state of a protein Purity evaluation of protein preparations

Local contacts: Gabriella Tedeschi (gabriella.tedeschi@unimi.it)

Quantitative studies of gene expression variation and relationships, detection of stages in cellular differentiation and studies

Analytical 2-D gel electrophoresis of surface associated proteins from S. aureus

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Western blotting

Technical Specifications: Mini Trans-Blot Cell from Biorad. Transfers two 7.5 x 10 cm gels Trans-Blot Cell from Biorad Transfers two 16 x 20 cm gels

Additional laboratory equipment Proteins blotted on nitrocellulose or polyvinylidendifluoride (PVDF) membranes can be further characterized by immunostaining or N-terminal amino acid sequencing

Fields of application Detection, identification and characterization of proteins following electrophoresis Differential quantitative analysis of expression or post-translational modification levels of proteins from different samples. Detection and characterization of new antigens

Evaluation of peripherin nitration in NGF treated PC12 cells

Probing for biologic activity of proteins, including protein-protein interactions

Local contacts: Gabriella Tedeschi (gabriella.tedeschi@unimi.it)

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Gel Image Analysis

The platform is equipped with a complete set of different gel image acquisition instruments and software allowing tailoring the analysis to specific customers’ needs. All postelectrophoresis staining conditions, including fluorescent dyes, or radioactive-labelled sample, can be detected and quantified over a large dynamic range

Supercooled high-resolution CCD camera with 1.44 megapixel resolution Real-time imaging 16-bit (65,535 gray level) data collection capability Choice of 3 UV transillumination wavelengths

Technical Specifications: 1) Molecular Imager GS-800 Calibrated Densitometer from Biorad Light source Fluorescent: white Wavelength: 400–750 nm Operating modes: Transmissive and reflective Sampling rate: 700 dpi Pixel density: 12-bit Resolution: 36.3 μm 2) Molecular Imager VersaDoc MP 4000 System from Biorad Excitation wavelengths: Red, green, blue, broadband UV, and white light Emission filters: 530BP, 605BP, 640BP, 695BP Detector: Front-illuminated highsensitivity CCD with microlens technology Multichannel image collection. Pixel size: 6.8 x 6.8 µm Pixel array size (H x V): 2,184 x 1,472 pixels Pixel data density: 16-bit (0–65,535 levels) Dynamic range: 3.4 orders of magnitude Illumination modes: Trans- and epiillumination 1) Molecular Imager ChemiDoc XRS from Biorad

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Software available: PD-Quantity One: a package for imaging and analyzing 1-D electrophoretic gels, dot blots, and western blots. Possibility to quantify and analyze a variety of data, including radioactive, chemilumenescent, fluorescent, and color-stained samples acquired from densitometers, storage phosphor imagers, fluorescent imagers, and gel documentation systems. PD-Quest: a package for analysis and databasing of 2-D gels. The software offers powerful comparative analysis tools to reveal subtle differences among gels being analyzed (for example, studying the effect of variables such as physiological/pathological conditions, dose-response, and time course).

Local contacts: Gabriella Tedeschi (gabriella.tedeschi@unimi.it)

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LC-ESI MASS SPECTROMETRY

Technical Specifications: 1) ThermoFisher LTQ Orbitrap Velos ETD Ion Max™ API source with S-lens ion optics technology Dual-Pressure Linear Ion Trap Orbitrap Mass Analyzer Atmospheric pressure ionization (API) ESI source and μESI source Nanospray source Proxeon Collision cell with axial field gradient Multiple fragmentation techniques: CID, HCD, and ETD Parallel MS and MSn analysis Resolving power of >100,000 Mass accuracy better than 1 ppm 2) Nano HPLC Ultimate 3000 Flow rate Nano 50-1000 nl/min Flow rate Cap 0.5-10 μl/min Flow rate Micro 10-160 μl/min Pressure range 0.1-50 MPa 6 eluent lines Biocompatible version DGP-3600MB (Titanium) Temperature range 5-85°C 2D HPLC possibility Autosampler WPS-3000

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Software available: Proteome Discoverer: comprehensive proteomics software including several database search engines to complement the breadth of dissociation techniques such as CID, ETD, and HCD for protein identification and PTM characterization, quantization and statistical analysis, automated data processing, Sieve: for automated, label-free semi-quantitative differential expression analysis of proteins,

peptides, and metabolites Bioworks: for protein identification Prosight: for top down identification and characterization of proteins, including their post-translational modifications (PTMs) Promass: for the analysis of intact proteins and oligonucleotides; optimized for high throughput applications

Local contacts:Gabriella Tedeschi (gabriella.tedeschi@unimi.it)

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MALDI TOF TOF MASS SPECTROMETRY

Technical specifications: Bruker MALDI TOF-TOF autoflex™ III * MALDI source scoutMTPTM with automatic loading sample Solid-state laser with SmartbeamTM technology, enabling MALDI imaging Linear flight tube (+ and – ions) of 120 cm Reflectron flight tube (+ and – ions) of 215 cm TOF/TOF technology (LIFT™) for fragmentations at high sensibility (LIDLIFT) and high energy (CID) PAN technology for bottom-up and topdown proteomics Patented AnchorChip™ MALDI target technology for fast automation, increased sequence coverage and 10–100 fold sensitivity increase over stainless steel holder

Software available: BioTools for protein data analysis FlexImaging for MALDI imaging

* The MALDI spectrometer is available at CIGA (University of Milano). Researchers of the proteomic platform have free access to this instrument

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Bruker disposable AnchorChip

Îą-Tubulin nitrated in rat brain

Fields of application for LC-ESI and MALDI mass spectrometry:

General proteomics including: analysis of the complete proteome of cells and tissues, protein identification by fingerprint and MS/MS analysis, molecular weight measurement, evaluation of protein expression, detection and characterization of posttranslational modifications, sequence analysis by MS/MS either by PSD, CID, HCD and ETD fragmentation techniques Shotgun LC-MS/MS analysis: direct analysis of complex mixtures to rapidly generate a global profile of the proteic components in the mixture. 2D-LC-MS and MS/MS analysis for the characterization of complex protein samples Label-free quantification of proteins and peptides based on mass spectrometry Clinical Proteomics including qualitative and quantitative differential proteomic

analysis of samples, new biomarker discovery and profiling, proteomics of disease and pathogens, evaluation of drug effects on protein patterns SERPA (serological proteome approach) analysis for the identification of antigens and development of new vaccines Network Mapping: identification of proteins in functional networks: biosynthetic pathways, signal transduction pathways, multi-protein complexes Food analysis by proteomics to authenticate food origin and quality including structural information on unknown ingredients of complex mixtures Polymers analysis: nucleic acids (DNA), peptide nucleic acids (PNA), block copolymers (nanostructures), functionalized polymers, macromolecules

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Tissue imaging: comprehensive visualization of the spatial distribution of biomarker candidates, drugs and metabolites in a tissue Analysis of low molecular weight compounds. The LC-MS/MS equipment allows to analyze samples previously separated on-line by LC (liquid chromatography). This approach allows the identification and characterization of low molecular weight molecules, including metabolites, drugs and their metabolites, xenobiotics and residues. It is particularly suitable to carry out

metabolic profiling to distinguish between normal and alterated states, to study mechanism of drug-induced toxicity and/or efficacy and to discover and identify potential biomarkers.

Local contacts: Gabriella Tedeschi (gabriella.tedeschi@unimi.it)

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Automated proteins/peptides sequencer

Technical specifications: Applied BioSystems Procise 491 automated protein/peptide sequencer for sequencing intact proteins and peptides based on Edman chemistry, starting from the N-terminal residue. Sample required: at least 40 pmol of protein which can be purified by conventional chromatographic methods or by electrophoresis followed by blotting on commercially available polyvinyliden difluoride (PVDF) membranes. Compatible with most chemicals used in protein science. Number of amino acids identified usually ranging from 10 to 20, depending on the starting material. Longer sequences can be obtained under favourable conditions.

Fields of application: Characterization of recombinant proteins: rapid determination of short N-terminal sequence of the intact protein of interest complements and confirms MS-based analyses for quality control of proteins obtained by recombinant-DNA technologies

longer stretches of amino acid from the Nterminus of proteins are frequently observed both as a result of post-translation modification of proteins from natural sources and during the overexpression and purification of recombinant proteins. This can result in microheterogeneities which must be identified for the complete characterization of the protein of interest. Characterization of proteins from organisms for which no genomic data are available. Edman chemistry always relies on a â&#x20AC;&#x153;de novoâ&#x20AC;? sequencing approach since assignment of the amino acid sequence from experimental results is independent from data-bank searches. Characterization of polypeptide patterns generated by limited proteolysis of the protein of interest to study domain organization, conformational changes and protein-protein interactions. Complementing MS-based determination of molecule weights and N-terminal sequence analysis of fragments separated by SDS-PAGE allows a complete description of the proteolytic pattern Local contact: Gabriella Tedeschi (gabriella.tedeschi@unimi.it)

Characterization of N-terminal processing of proteins: removal of N-terminal Met or of

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Amino acid analysis

Technical specifications: Japan Spectroscopy (Jasco) HPLC equipped with PU980 pumps and 821-FP spectrofluorometer detector. Method: pre-column derivatization of primary amino acids with orthophthalaldehyde (OPA) followed by RPHPLC separation and fluorescence detection

-Exact determination of the protein content of a sample (with a precision unparalleled by any other general method)

Local contact: Gabriella Tedeschi (gabriella.tedeschi@unimi.it)

Sample amount required: the methods routinely allows quantifying a minimum of 10 pmol of each amino acid, although sensitivity can be lowered but requires careful preliminary handling and preparation of samples by the subject requiring the analysis. Standard error: Âą 5 % Determination of specific non-proteic amino acids (for example for the metabolic profiling of free amino

acids in blood) can be performed but may require additional set up of chromatographic conditions for the separation of such amino acids

Fields of application: All studies requiring the determination of the exact amino acid content of a sample, such as: -Metabolic profiling of biological fluids such as blood -Amino acid content of food

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SURFACE FUNCTIONALIZATION THIN FILMS DEPOSITION Cleanrooms

The Micro- and Nano-fabrication platform is equipped with a 70m2 environment with a controlled level of dust contamination. Different rooms with different contamination levels (between ISO8 and ISO5) are present. A photolithography room with suitable yellow lights is available. Local Contacts: Gero Bongiorno (gero.bongiorno@fondazionefilarete.com)

Spin coater model WS650Sz-6NPP-Lite from Laurell Technologies Corporation

Capability to coat up to 6â&#x20AC;? diameter (150mm) wafer or 4â&#x20AC;? (100mm) square substrates. Standard holders for microscope slides and holders for fragments with dimensions and shapes down to 10mm squares.

Local Contacts: Gero Bongiorno (gero.bongiorno@fondazionefilarete.com)

The instrument is equipped with a Digital Processor Controller and remotely connected to a PC. Rotation speed between 100 and 12000 RPM (Resolution > 0.5RPM). Selectable acceleration up to 13KRPM/sec. Unlimited programs storage capability. Each program can consist in up to 51 steps. Double automatic dispensing system (one of which is orientable in order to allow the coating of the outer part of the spinning wafer).

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NanoOnSi nanostructured thin films deposition system from Tethis

NanoOnSi is a system for the deposition of thin films of nanostructured materials by means of the so called Supersonic Cluster Beam Deposition technique (SCBD). It is composed of two vacuum chambers: the expansion chamber for the generation of the supersonic beam and the deposition chamber for the collection of nanoparticles on substrates. The apparatus is equipped with a PMCS™ (Pulsed Microplasma Cluster Source) that is able to produce metal and metal-oxides nanoparticles. A robotic holder for handling the substrates is placed in the deposition chamber. The system is able to omogeneously coat surfaces up to 200x200mm 2 big. The system is interfaced to the cleanroom facility allowing the production of thin films in highly clean environment. · Typical rate data: 100 nm homogeneously deposited on a 10 cm2 surface in 1 minute. · Deposition thickness from sub-monolayers dispersed nanoparticles up to several microns can be achieved. · Real time control of the thin film growth through quartz balances. · Room temperature process allows the use of every kind of substrate (metals, ceramics, polymers, membranes, micro-electro-mechanical systems). · Patterning with sub-micrometric lateral resolution can be achieved by simple non-contact stencil mask method. The materials produced have found applications in the following fields: DEVICES · · · ·

Nanoporous Oxide Films for Conductimetric Gas Sensing Nanoporous Noble-Metal Films for Cathalitic Gas Sensing Nanoporous Oxigen/Water Reactive Layers for Gas-gettering Nanostructured Gold Films for Thiol adsorption in Bio-Sensors

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CATALYSIS · Nanoporous Noble-Metal Thin Films for PEM Fuel Cells · Nanoporous Photo-Catalytic Layers · Dispersed Nanostructured Catalyst for Nanotubes Growth ENERGY · Nanoporous Electrodes in ECDL Supercapacitors and Batteries · Nanoporous Electrodes in Dye-Sensitized Photovoltaic Cells BIOTECH · Bio-compatible Layers for Lab-On-Chip Applications · Bio-compatible Layers for DNA-Protein-Virus-Bacterial-Cell Arrays Local Contacts: Gero Bongiorno (gero.bongiorno@fondazionefilarete.com)

TECNA box coater for thin films deposition mounting a Ferrotec e-beam evaporation source. This system is specifically designed for the deposition of multilayered thin films using an e-beam evaporation source. The system is interfaced to the cleanroom facility allowing the production of thin films in highly clean environment. This system is able to manage substrates with diameters up to 10mm. Thanks to an IR furnace, the substrate temperature can be risen up to 600°C. The pressure during the deposition process can be as low as 10-8mbar. The heart of the system is the Ferrotec EV-M6 multipocket e-beam source. This source is able to deposit sequences of up to 4 different materials (metals or metal oxides) and is suitable for a wide range of applications including semiconductor, thin film and optical coating.

Local Contacts: Gero Bongiorno (gero.bongiorno@fondazionefilarete.com)

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Plasma System Colibrì from Gambetti

This is a simple and easy to use desk-top plasma system, designed for those needing to clean, modify or activate surfaces of metal, plastic, ceramic or paper-based materials and more. Ideal for R&D, or for small productions. The power supply works in RF regime at 13,56 MHz, 200W. The plasma chamber is cylindrical with a diameter of 100mm and a depth of 260,5mm. The system is controlled by a microcomputer and uses two mass flow-controllers (MFC) for the regulation of the percentage of a single gas, or to define the ratio of two different gases.

Local Contacts: Gero Bongiorno (gero.bongiorno@fondazionefilarete.com)

Applications : · · · ·

Surface activation Surface cleaning Surface coating Etching

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OTHER INSTRUMENTS Agilent 2100 Bioanalyzer for electrophoresis The Agilent 2100 Bioanalyzer is a microfluidics-based platform for sizing, quantification and quality control of DNA, RNA (including miRNA), proteins and cells on a single platform. Results (10 to 12 samples per run) are delivered within 30-40 minutes in automated, high quality digital data, making this instrument faster and more reliable than a gel. Among the advantages of using this instrument are for examples: · · · · · ·

Ready-to-use assays and pre-packaged reagent kits Reproducible data, contamination-free hardware Quick and easy sample comparison Reproducible, complete and digital data Easy handling, storage and exchange of digital data Quantification of each individual fragment per sample offers purity results 2-color analysis of fluorescently stained cells

This instrument exploits the “Lab-on-a-Chip” technology utilizing a network of channels and wells that are etched onto polymer chips to build mini-labs. The main advantages of Lab-on-a-Chip are ease-of-use, speed of analysis, low sample and reagent consumption and high reproducibility due to standardization and automation. Each chip contains an interconnected set of gel-filled channels that allow for molecular sieving of nucleic acids or protein samples. A series of electrodes control sample movement within the chip. These make contact with the samples when the instrument lid is closed. Each electrode is connected to an independent power supply, providing maximum control and flexibility. The electrode cartridge is also removable, providing the flexibility to implement different configurations depending on the design of a chip

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LI-COR LI-6400XT gas Exchange system

The LI-6400XTA is designed for molecular biologists, plant physiologists, and geneticists who study Arabidopsis thaliana. It provides a platform for physiological assessment of in situ function to validate regulatory or functional genes identified by genomic, molecular, or bioinformatics studies.

Key Features · Robust, reliable gas analyzers for rigorous field use. · Non-dispersive infrared analyzers with 0 - 3000 ppm CO2, 0 - 75 ppth H2O ranges. · Gas analyzers in the sensor head provide rapid response and eliminate time delays. · User cleanable optical path - saves time and money · Gold plated mirrors provide long term stability · 64 MB internal flash memory and removable compact flash memory card for data storage. · External connection to Local Area Network. · Adjustable contrast, backlit LCD graphic display. · Eight independent graphics screens quickly toggle between graphic and numeric screens.

Photosynthesis The LI-6400XT is the only photosynthesis measu-rement system to put the CO2 and H2O gas analyzers in the sensor head. These dual path, non-dispersive infrared analyzers feature an open path design with the optical bench of the sample analyzer open directly to the leaf chamber mixing volume. Leaf dynamics are measured in real time, preventing confounding correlations between gas exchange and changes in environmental driving variables. Whole Plant Arabidopsis Gas Exchange System

Is a versatile tool, designed to provide scientists with the flexibility to answer a wide variety of research questions. Interchangeable chamber bottom plates permit measurement of plants grown in either pots or Cone-tainers™. With the RGB Light Source, which enables light response studies of whole rosettes, researchers can select any combination of red, green, or blue light with continuously variable intensities, or white light by using equal proportions of red, green, and blue light. Low temperature LEDs with an attached Peltier cooler minimize the influence of the light source on the temperature of the sampling volume and plant, while a silicon feedback photodiode ensures accurate lighting intensity for the duration of measurements. The RGB Light Source mounts directly to the WPA Chamber to provide uniform light distribution for every measurement.

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Integrated Fluorescence Measurements 6400-40 Leaf Chamber Fluorometer

The 6400-40 Leaf Chamber Fluorometer transforms the LI-6400XT System into the most integrated Portable Fluorescence and Gas Exchange System available. Field-installable, the fluorometer easily and quickly attaches to the LI-6400XT sensor head. Simultaneous measurement of gas exchange and fluorescence over the same leaf area. Complete control of the leaf environment for collection of gas exchange and fluorescence data from a single, portable unit. User-defined manual or automatic measurement protocols.

The Leaf Chamber Fluorometer is a pulseamplitude modulated (PAM) fluorometer that can be used to take measurements on both dark- and light-adapted samples. Measured parameters include Fo, Fm, F, Fm', and Fo', and calculated parameters include Fv, Fv/Fm, dF/Fm, qP, qN, NPQ, and ETR. The 6400-40 provides complete control over the actinic and saturation (independently controlled red 630 nm and blue 470 nm LEDs), measuring (red 630 nm LEDs, modulated from 0.25 to 20 kHz), and far-red (740 nm LED for PSI excita-tion) light. The unique design of the 6400-40 Leaf Chamber Fluorometer eliminates the need for fragile, awkward fiber optic light guides. Lightweight design and low power consumption make it possible for one person to gather data quickly and easily. Calibration information for the Leaf Chamber Fluorometer is stored onboard, making it easy to move between different LI-6400XT consoles. Extended Reach Chamber This design allows you to access and measure small leaves that are tightly bunched together

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BTX ECM 830 Square- wave electroporator Overview Electroporation, or electropermeabilization, enables the formation of pores in cell membranes caused by an externally applied electrical field. It is usually used in molecular biology as a way of introducing some substance into a cell, such as loading it with a molecular probe, a drug that can change the cell's function, or a piece of coding DNA. Laboratory equipment: BTX's ECM 830 Electroporator is a Square Wave Electroporation System designed for all in vitro and in vivo electroporation.The ECM 830 possesses key features including finer voltage discrimination, Arc Quenching, the monitoring of all key parameters, and the control of pulse intervals. Indeed, the ECM 830 is capable of generating 1-500V in low voltage (LV) mode and 03000V in high voltage (HV) mode. The pulse length range is 10microsec to 1sec in LV mode and 10us to 600us in HV mode. You can set up to 99 pulses at 0.1 to 10 pulses per second frequency.

The ECM 830 Electroporator is equipped with 3 specialty electrode to realize different applications:

¡ BTX Electroporation Cuvettes Plus are designed for use in electroporation and electrofusion of bacteria, yeast, insect, plant and mammalian cells. The cuvettes are molded with embedded aluminum electrodes, washed prior to packaging and gamma irradiated for guaranteed sterility. Three electrode gap sizes are available, 4mm, 2mm and 1mm for high field strengths up to 25.0 kV/cm. ¡ The Petri Pulserâ&#x201E;˘ PP35-2P is a reusable electroporation applicator designed to fit into each single well of a 6-well plate or an individual 35mm diameter petri dish. The PP35-2P consists of an electrode assembly embedded in a polyurethane holder with high voltage

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electrode cables. The thin electrodes are gold plated and designed to maximize the surface area of electroporation. The Petri Pulser PP35-2P is designed for the electroporation of adherent cells in situ or as an alternative to cuvette electroporation for larger volumes or multiple cell samples. The use of the electrode for the electroporation of adherent cells in situ eliminates the need for trypsinization or mechanical removal of cells from their growth substrate. · BTX Tweezertrodes are reusable, tweezer-style in vivo electrodes for drug or gene delivery in animals. Following localized or systemic injection of the molecule of interest, the Tweezertrode electrode disks are used to grasp the tissue of interest. An electroporation pulse is then given, initiating pore formation and incorporation of the molecule into the cells of the tissue in direct contact with the electrode disks Applications: · Mammalian Cell Protein/Drug Electroincorporation · Mammalian Cell Transfections/Gene Therapy · Ex-utero and In-utero Electroporation · Plant, Yeast and Bacterial Electroporation Application

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CONTACT

TO ORDER/FOR INFORMATION

services@fondazionefilarete.com

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9 technological platforms services: Genomics and bioinformatics, proteomics, CNS cell model, Stem Cell, Cellular imaging, Animal Model, Path...