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Purification of cells from human peripheral blood • Mononuclear cells: Lymphocytes + Monocytes • Monocytes • Polymorphonuclear leukocytes (PMNs) or granulocytes • Platelets or thrombocytes


Density of human blood cells Cell number

1.060

1.070

Monocytes Lymphocytes Basophils Neutrophils Eosinophils Erythrocytes

1.080

1.090

1.100 Density (g/ml)

Mononuclear Polymorphonuclear leukocytes


Isolation of human mononuclear cells I Density barrier method

600g 15 min 1.077 g/ml

MC RBC PMNs


Axis-Shield Density Gradient Media for isolation of human PBMCs • Lymphoprep™: 9.1% diatrizoate, 5.7% polysaccharide; 1.077 g/ml, 295mOsm; endotoxin <0.13 EU/ml • Nycoprep™ 1.077: 14.1% Nycodenz®, 0.44% NaCl, 5 mM Tricine-NaOH, pH 7.0; 1.077 g/ml, 295 mOsm; endotoxin < 0.13 EU/ml • Optiprep™ (endotoxin < 0.13 EU/ml) diluted with any suitable balanced salt solution or culture medium to give a 1.077 g/ml medium (C03)


Separation of PBMCs on Lymphoprep™ Blood:saline 1:1 Blood < 12 h old

6 ml diluted blood on 3 ml of Lymphoprep™


Lymphoprep™ competitors • Ficoll-Paque (GE Health Care (ex Amersham and Pharmacia) • Histopaque 1077 (Sigma) • Not endotoxin tested • More expensive endotoxin-tested versions, e.g. Sigma’s Histopaque 1077 Hybrimax


Lymphoprep™ queries (I) • • • • • • • • •

Anticoagulant? Not anticoagulant sensitive Do I have to dilute the blood? Whole blood less easy to layer; poorer % yields Can increase sample:Lymphoprep™ ratio Yes – but not recommended Can I use a leukocyte-rich plasma? Yes, OK to separate PBMCs and PMNs Centrifugation at 4°C rather than room temperature? • Yes, but need to increase time by 5 min


Lymphoprep™ queries (II) • • • • • • • • •

Ficoll-Paque or Histopaque work better Impossible – media have identical composition Poor definition of PBMC band at interface Poor layering technique and/or brake problem Poor separation from erythrocytes/PMNs Blood > 12 h old; clinical specimens Can I use it for non-human blood? Maybe primates and some ruminants How can I remove the platelets?


Lymphoprepâ&#x201E;˘ Tube Blood diluted 1:1 with saline 800g for 15 min Plastic frit LymphoprepTM

Medium displaced upwards

PBMCs


Isolation of PBMCs by flotation mixer method (C04) 10 ml blood saline

1.25 ml OptiPrep

1500g

PBMCs

30 min

Plasma + Platelets PMNs + RBCs


Problem with mixer method • • • •

Ratio of cells:plasma (haematocrit value) Normal males: 42-54% Normal females: 38-46% Some clinical samples may have significantly lower values


Removal (isolation) of platelets on a density barrier (C12) 5 ml whole blood 5 ml of 1.063 g/ml

350g 15 min Platelets Leukocytes + RBCs


Isolation of platelet-free PBMCs (C05) saline

PBMCs 600g

1.077 g/ml Blood + OptiPrep 1.095 g/ml

20 min PMNs Plasma + RBCs + Platelets


Axis-Shield Density Gradient Media for the isolation of human PMNs from whole blood • • • •

Polymorphprep™ 13.8% diatrizoate, 8.0% dextran 500 Density = 1.113 g/ml Osmolality = 460 mOsm


Separation of PBMCs and PMNs on Polymorphprepâ&#x201E;˘ 5 ml

Whole blood 450-500g 30-35 min at 20oC

5 ml

Polymorphprep

PBMCs PMNs RBCs


How does Polymorphprepâ&#x201E;˘ work? 1

2

3

4


Polymorphprepâ&#x201E;˘ Separation


Relative number

Coulter STKR Analysis of PBMCs and Polymorphs

4

8

12

16

20

24

28

Cell vol (femtolitres x 0.1)

32


Polymorphprep – critical points • • • • • • • •

Must use fresh (<2 h old) whole blood Anticoagulant must be EDTA (dipotassium) Temperature must be 18-22°C Blood:medium volume ratio must be 1:1 5 ml + 5 ml in a 15 ml tube Brake must NOT be used Centrifugation time can be + 5 min Recommended g-force is the gav


Geometry of rotors rmax rav rmin axis of rotation

Set rpm speed to give 500g at rmax Equivalent to 400g at rav


Polymorphprep/sample volumes


Human PMNs from a leukocyterich plasma (C011)

600g 20 min

PBMCs

1.077 g/ml 1.10 g/ml

PMNs


Axis-Shield Density Gradient Media for the isolation of human monocytes: barrier sedimentation (C55) • 13.0% Nycodenz, 0.58% (w/v) NaCl, 5 mM Tricine-NaOH, pH 7.0 • Density = 1.068 g/ml • Osmolality = 335 mOsm


Density of human blood cells Cell number

1.060

1.070

Monocytes Lymphocytes Basophils Neutrophils Eosinophils Erythrocytes

1.080

1.090

1.100 Density (g/ml)


Separation of monocytes on 335 mOsm, 1.068 g/ml barrier LRP 600g 15 min 1.068 g/ml

M L

Harvest zone


Nycoprep™ 1.068 requirements • • • • • •

Leukocyte-rich plasma; not whole blood Anticoagulant: EDTA Blood from normal individual, < 2 h old Room temperature 18-22°C No brake to decelerate rotor Centrifugation conditions may need optimizing from lab to lab


Flotation of monocytes from leukocyte-rich plasma (C09) HBS

M 600-800g

1.068 g/ml

15-20 min

L

1.084 g/ml

LC LRP

P


FACS analysis of monocyte band Graziani-Bowering, G.M. et al (1997)J. Immunol. Meth., 207, 429-436

Surface Marker

% events

CD3+

3.4

CD14+/CD4â&#x20AC;&#x201C;

1.6

CD14â&#x20AC;&#x201C;/CD4+

6.9

CD14+/CD4+

84.1

All monocyte markers

92.6


Monocyte flotation isolation queries • • • •

No monocyte band observed Band may be quite diffuse Poor recovery and purity of monocytes Rapid preparation of leukocytes essential and handling of cells must be very gentle • Does method work at 4°C? • Probably


Axis-Shield Density Gradient Media: isolation of rodent PBMCs, sedimentation on to a barrier (C43) • Dilute buffered saline with water (2.5:0.5) Solution is 242 mOsm • Dilute OptiPrep with the 242 mOsm solution (2.7: 9.3) • Solution of 1.077 g/ml and 265 mOsm • Competition (Histopaque 1.083): 1.083 g/ml, 295 mOsm


Axis-Shield Density Gradient Media for the isolation of rodent PBMCs Cell number

1.060

1.070

Monocytes Lymphocytes Basophils Neutrophils Eosinophils Erythrocytes

1.080

1.090

1.100 Density (g/ml)


Rodent PMNs from leukocyte-rich plasma (C45) Aspirate 600g 20 min

LRP MC 1.077 g/ml 265 mOsm RBC + PMNs

Isotonic ammonium chloride


Common flotation strategy for purifying low density cells from tissues • Isolation of dendritic cells from spleen, thymus, lymph nodes etc (C20) • Tissue disaggregated using enzymes • Cell suspension adjusted to 1.085 g/ml • Make up solution of 1.065 g/ml


Dendritic cells â&#x20AC;&#x201C; flotation strategy Reudl, C. et al (1996) Eur. J. Immunol., 26, 1801-1806 Saline or culture medium 1.065 g/ml

Sample 1.085 g/ml

Dendritic cells 600g 15 min


Pancreatic islets – flotation (C18) Van der Burg, P.P.M. (1998) Transpl. Proc. 30, 362-363 OptiPrep H’paque 1.09 g/ml 1.10 g/ml

Islets

Yield

92%

58%

500g/5 min

Purity

86%

61%

Viab’lty

75%

54%

Acinar cells


Removal of non-viable cells (C13) Culture medium 1.12 g/ml 800g 20-25 min Cell suspension adjusted to 1.15-1.16 g/ml

Viable cells


Other Axis-Shield Cell Applications • • • • • • • •

Epithelial cells from gastric mucosa (C28) Neurons: spinal cord (C22) brain (C29) Human erythrocytes and reticulocytes (C35) Megakaryocytic progenitor cells (C23/C48) Pneumocytes and other lung cells (C25/C44) Stellate cells from liver and pancreas (C33) Hepatic Kupffer cells (C50) Renal cells (C42)


Publications database on cells • • • • •

OptiPrep (since 1994) over 650 Nycodenz® (since 1984) approx 2000 Using either the Applications CD or the website: www.axis-shield-density-gradient-media.com Follow the instructions to access the relevant Index • Click on the cell of interest


immunology