IncuCyte® Application Posters A decade of development of live-cell analysis Posters Cell Health and Quality Control
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Cell Migration, Invasion, and Chemotaxis
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Immune Cell Functions—Cell Killing and Engulfment
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Differentiation Assays—Neurite Outgrowth and Angiogenesis
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IncuCyte® systems throughout the years
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n 2004, Kirk Schroeder and Brad Neagle of Essen BioScience contemplated an empirical and troubling observation that expression levels of hERG K+ channels in recombinant cells were profoundly sensitive to changes in cell confluence. If only they could measure, predict and control the confluence of their cultures then they could significantly improve the quality of their hERG electrophysiology assays. One year on, they had developed the first system that could automatically measure cell confluence, non-invasively, in realtime, from directly inside an incubator. And so, IncuCyte® live-cell analysis was born. Over a decade later, IncuCyte live-cell analysis has evolved as a frontline and enabling method not only for cell QC and monitoring but also advanced phenotypic assays. A wide range of specific applications have been developed, variously comprising reagents, software, hardware and protocols. In this e-book, we illustrate this through a compilation of scientific posters that have been presented at congresses and meetings over the years. Validation experiments in relevant biology model systems are described to exemplify real-world use and the key value statements for this method. Together, these posters highlight the breadth of applications as well as the biology problem-based, ‘integrated-solution’ approach that has driven the evolution of real-time live-cell analysis.
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Cell Health and Quality Control
Posters 1–7
Cell monitoring and growth and viability assays over time are the ‘stock-in-trade’ of IncuCyte®. Building on the initial phase-contrast confluence measures in 2D adherent cell cultures, a suite of image capture modalities and analyses have been developed to extend this use. For example, whole-well imaging enabled rare-event capture in large area dishes (e.g. for stem cell reprogramming - see Poster 1), while at the other end of the spectrum learnings in cell handling and plating allowed miniaturization of growth assays into 384-well format (Poster 2). Tumor spheroid assays in round-bottomed ULA plates were made possible via novel auto-focusing and analysis
algorithms and the introduction of bright-field imaging (Posters 3 & 4). In another example, the unique mobile optical system design of IncuCyte® has been exploited for assays on stationary, non-adherent cells (Poster 5). The introduction of specialized non-perturbing fluorescent reagents has been particularly valuable for cell counting and cell health assays, both in mono- and coculture model systems. These reagents fall into two broad categories – fluorescent proteins and dyes for labelling cells (e.g. NucLight & CytoLight) and fluorescent reporters for cell death and apoptosis (e.g. Cytotox, Caspase 3/7 and Annexin V fluorogenic dyes – Posters 6 & 7). Page
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Capturing rare events in iPSC reprogramming using live cell analysis
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Miniaturized live-cell kinetic imaging assays in 384-well format
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Simplifying high throughput 3D spheroid growth and shrinkage assays using live cell analysis
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Live-cell analysis of 3D spheroids: label-free & fluorescent cell health reporters
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Continuous live-cell proliferation, clustering and viability assays for T-cells, PBMCs, monocytes and B-cells
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Multiplexed, live-cell analysis enabled: reagents, assays and IncuCyte® Zoom
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Validation of IncuCyte® live-cell labelling dyes for monitoring immune and tumour cell interactions INCUCYTE® APPLICATION POSTERS
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Poster 1
Cell Health & Quality Control:
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Poster 2
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Cell Health & Quality Control:
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Poster 3
Cell Health & Quality Control:
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Poster 4
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Poster 5
Cell Health & Quality Control:
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Poster 6
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Cell Health & Quality Control:
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Poster 7
Cell Health & Quality Control:
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Cell Migration, Invasion, and Chemotaxis The development of live-cell analysis solutions for measuring cell movement typifies the ‘integrated turnkey’ approach that we have adopted. Poster 8 describes validation data for a kinetic ‘scratchwound’ assay comprised of a mechanical pin ‘Woundmaker’ tool, an ImageLock 96-well plate, and a custom software module for automatically acquiring and analyzing wound images. When used together these components deliver a facile, flexible and powerful assay for cell migration. By plating cells in a bio-matrix sandwich layer (e.g. collagen, Matrigel) the solution can be extended to measurements of cell invasion (Poster 9).
Posters 8–11
Similarly, for chemotaxis studies, a novel 96-well trans-well plate consumable (ClearView™) has been developed that allows visualization of cells migrating toward chemo-attractants, via precision laser-drilled holes in an optically clear membrane. When combined with new analysis algorithms, researchers are now able to automatically quantify the full time-course of the movement of cells from the top to the under-side of the membrane (Poster 10). Poster 11 illustrates how these and other assays can be deployed in the cancer biology research area.
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High-fidelity 96-well kinetic imaging assays for cell migration
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Differential biology of tumor cell migration and invasion through bio-matrices measured with 96-well live-cell kinetic imaging assays
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IncuCyte® Chemotaxis System: a new and enabling solution for directional migration assays
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Live-cell analysis of 3D spheroids: label-free & fluorescent cell health reporters
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Cell Migration, Invasion, Chemotaxis
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Poster 8
Poster 9
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Cell Migration, Invasion, and Chemotaxis
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Poster 10
Poster 11
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Immune Cell Functions— Cell Killing and Engulfment Live-cell analysis has proven particularly powerful for understanding more advanced cell systems, where the temporal and spatial interactions between cells are critical to the phenotypic outcomes. This is nicely exemplified by the immune-cell killing application (Posters 12, 13 and 14). Here, fluorescent cell labels and/or apoptosis/cell health reporter dyes are used to quantify the killing of tumor cells by activated immune cells (e.g. T-cell, Natural Killers) over time. As an important validation, immune cells can be clearly observed to attack the tumors and induce the morphological hallmarks of apoptosis (e.g. nuclear condensation, cell shrinkage, loss of motility). This format has been validated for a variety of immune-cancer cell paradigms including adherent and non-adherent target
Posters 12–27
cells, monolayer and spheroid models as well as ADCC and CDC assays. A second important function of the immune system is clearance of foreign proteins and dying cells. Using pH-sensitive fluorophores coupled to bacterial proteins we have assembled real time assays for phagocytosis that report internalization of the conjugate into the acidic phagolysosome (Poster 15). The same principle has been used to monitor engulfment of apoptotic neutrophils and tumor cells (‘efferocytosis’ – Poster 16). Poster 17 describes how different live cell assays can be combined to address important questions in immunotherapy.
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Novel kinetic live cell imaging assays for T cell killing of tumour cells
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Validation of novel continuous live-cell assays for immune cell activation and killing of blood cell cancers
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96-well live-cell assays for immune cell killing of 3D tumour spheroids
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Real time image-based quantification of phagocytosis in living cells using IncuCyte® ZOOM
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CD47 antibody-induced engulfment of human leukaemic T-cells by bone-marrow derived macrophages
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Quantitative live-cell imaging assays for immunotherapy: chemotaxis, immune cell killing & phagocytosis
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A DECADE OF DEVELOPMENT OF LIVE-CELL ANALYSIS
Poster 12
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Immune Cell Functions — Cell Killing and Engulfment
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Immune Cell Functions — Cell Killing and Engulfment
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Poster 13
Poster 14
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Immune Cell Functions — Cell Killing and Engulfment
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Immune Cell Functions — Cell Killing and Engulfment
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Poster 15
Poster 16
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Immune Cell Functions — Cell Killing and Engulfment
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Immune Cell Functions — Cell Killing and Engulfment
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Poster 17
Posters 18–22 Differentiation Assays– Neurite Outgrowth and Angiogenesis Continuing the theme of advanced cell models, we have developed turnkey solutions for measuring vascular network formation (angiogenesis) and neurite dynamics in human co-culture systems. Again, these combine new cellular reagents and protocols with specialized yet simple to use analysis software. For angiogenesis, two different cell kits have been optimized: (1) a 10-day primary cell model with GFPlabelled human vascular endothelial cells and (unlabeled) dermal fibroblasts in co-culture, and (2) a 2-4 day mesenchymal stem cell model with GFP-endothelial
colony forming cells and (unlabeled) adipocyte derived stromal cells. In each case, the endothelial cells form vascular networks that are automatically quantified via the green fluorescence signal (Posters 18 & 19). Neurite dynamics can be measured in neurons in monoculture via the phase-contrast image, or preferably in co-cultures with astrocytes or glial cells. For co-culture, IncuCyte® NeuroLight Red is used to express a membranetargetted mKate2 fluorescent protein via a neuronalspecific synapsin promotor (Posters 20-22).
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Comparative morphological, temporal and pharmacological profiles of two 96-well in vitro kinetic angiogenesis co-culture assays
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Assembly and pharmacological validation of a fully kinetic 96-well in vitro vascular tube formation assay
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Quantifying neurite dynamics in human iPSC derived neuronal mono- and co-cultures using long term live-cell imaging
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Kinetic and label-free, live-cell analysis assays for neurite outgrowth in primary, iPSC-derived and immortalized neurons
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Quantitative Live-cell Analysis for Optimization of Culture Conditions and Evaluation of Cell Health In Human Induced Pluripotent Stem Cell-derived Neurons
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Differentiation Assays—Neurite Outgrowth and Angiogenesis
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A DECADE OF DEVELOPMENT OF LIVE-CELL ANALYSIS
Poster 18
Poster 19
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Poster 20
Poster 21
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Poster 22
Perspective The biological applications and datasets covered in this poster set illustrate the power, breadth and versatility of live-cell analysis in biomedical research. This work is further substantiated by the publication of >1000 independent and peer-reviewed scientific papers that contain IncuCyte® data: essenbioscience.com/en/resources/publications/ From this, the key, application-independent, value statements of live-cell analysis are demonstrated: (1) greater biological insight afforded by observing and quantifying cells non-invasively and in real-time (2) productivity gains arising from simple workflows and parallelized automated data capture and analysis. At the level of specific applications, the provision of turnkey and fully integrated solutions further enhances researcher’s output. Going forward, and as before, further developments of IncuCyte® live-cell analysis will be biology problem driven and directed toward the unmet needs of the cell biologist.
Contributors and Acknowledgements The science presented in this e-book is a product of the sustained, multi-disciplinary R&D activities of Essen BioScience personnel based in Ann Arbor, Michigan USA and Welwyn Garden City, Hertfordshire UK over the last decade. This includes cell biologists, software engineers, optical and hardware designers as well as essential back up support functions. Some of the work was done in collaboration with external partner companies.
Individual names have been removed from posters not to discredit them, rather to recognize the contribution of the entire Essen team, both past and present. You know who you are. A huge debt of gratitude is due—this would not have been possible without you. We press on together in our mission of delivering innovative and game-changing solutions to further human health.
The posters in this compendium represent research performed during the last 10 years, using products available at the time of creation. Accordingly, our product strategy and portfolio have evolved. Please note that there may be products cited that are obsolete or unavailable for purchase. © 2017 Essen BioScience. All rights reserved. IncuCyte®, Essen BioScience® and all names of Essen BioScience products are registered trademarks and the property of Essen BioScience unless otherwise specified. Essen Bioscience is a Sartorius Company.
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