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

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Presentation on theme: "Purification of cells from human peripheral blood Mononuclear cells: Lymphocytes + Monocytes Monocytes Polymorphonuclear leukocytes (PMNs) or granulocytes."— Presentation transcript:

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

2 Cell number Density (g/ml) Monocytes Lymphocytes Basophils Neutrophils Eosinophils Erythrocytes Mononuclear Polymorphonuclear leukocytes Density of human blood cells

3 Isolation of human mononuclear cells I Density barrier method MC PMNs RBC 600g 15 min g/ml

4 Axis-Shield Density Gradient Media for isolation of human PBMCs Lymphoprep™: 9.1% diatrizoate, 5.7% polysaccharide; g/ml, 295mOsm; endotoxin <0.13 EU/ml Nycoprep™ 1.077: 14.1% Nycodenz®, 0.44% NaCl, 5 mM Tricine-NaOH, pH 7.0; 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 g/ml medium (C03)

5 Separation of PBMCs on Lymphoprep™ Blood:saline 1:1 Blood < 12 h old 6 ml diluted blood on 3 ml of Lymphoprep™

6 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

7 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

8 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?

9 Lymphoprep™ Tube Blood diluted 1:1 with saline 800g for 15 min PBMCs Medium displaced upwards Lymphoprep TM Plastic frit

10 Isolation of PBMCs by flotation - mixer method (C04) 1500g 30 min saline 1.25 ml OptiPrep 10 ml blood PBMCs PMNs + RBCs Plasma + Platelets

11 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

12 Removal (isolation) of platelets on a density barrier (C12) 350g 15 min Platelets Leukocytes + RBCs 5 ml of g/ml 5 ml whole blood

13 Blood + OptiPrep g/ml g/ml saline 600g 20 min Isolation of platelet-free PBMCs (C05) PBMCs PMNs Plasma + RBCs + Platelets

14

15 Polymorphprep™ 13.8% diatrizoate, 8.0% dextran 500 Density = g/ml Osmolality = 460 mOsm Axis-Shield Density Gradient Media for the isolation of human PMNs from whole blood

16 g min at 20 o C Whole blood 5 ml Polymorphprep 5 ml PBMCs PMNs RBCs Separation of PBMCs and PMNs on Polymorphprep™

17 321 How does Polymorphprep™ work? 4

18 Polymorphprep™ Separation

19 Coulter STKR Analysis of PBMCs and Polymorphs Cell vol (femtolitres x 0.1) Relative number

20 Polymorphprep – critical points Must use fresh (<2 h old) whole blood Anticoagulant must be EDTA (dipotassium) Temperature must be  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 avRecommended g-force is the g av

21 Geometry of rotors axis of rotation r max r av r min Set rpm speed to give 500g at r max Equivalent to 400g at r av

22 Polymorphprep/sample volumes

23 Human PMNs from a leukocyte- rich plasma (C011) PBMCs PMNs 600g 20 min g/ml 1.10 g/ml

24 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 = g/ml Osmolality = 335 mOsm

25 Cell number Density (g/ml) Monocytes Lymphocytes Basophils Neutrophils Eosinophils Erythrocytes Density of human blood cells

26 Separation of monocytes on 335 mOsm, g/ml barrier g/ml LRP 600g 15 min M L Harvest zone

27 Nycoprep™ 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

28 Flotation of monocytes from leukocyte-rich plasma (C09) g min HBS g/ml g/ml LRP LC M L P

29 FACS analysis of monocyte band Graziani-Bowering, G.M. et al (1997)J. Immunol. Meth., 207, Surface Marker% events CD3+3.4 CD14+/CD4–1.6 CD14–/CD4+6.9 CD14+/CD All monocyte markers92.6

30 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

31 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 g/ml and 265 mOsm Competition (Histopaque 1.083): g/ml, 295 mOsm Axis-Shield Density Gradient Media: isolation of rodent PBMCs, sedimentation on to a barrier (C43)

32 Cell number Density (g/ml) Monocytes Lymphocytes Basophils Neutrophils Eosinophils Erythrocytes Axis-Shield Density Gradient Media for the isolation of rodent PBMCs

33 Isotonic ammonium chloride Rodent PMNs from leukocyte-rich plasma (C45) MC RBC + PMNs 600g 20 min Aspirate g/ml 265 mOsm LRP

34 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 g/ml Make up solution of g/ml

35 Dendritic cells 600g 15 min g/ml g/ml Sample Saline or culture medium Dendritic cells – flotation strategy Reudl, C. et al (1996) Eur. J. Immunol., 26,

36 Pancreatic islets – flotation (C18) Van der Burg, P.P.M. (1998) Transpl. Proc. 30, g/5 min Islets Acinar cells 1.09 g/ml 1.10 g/ml OptiPrepH’paque Yield92%58% Purity86%61% Viab’lty75%54%

37 Removal of non-viable cells (C13) 800g min Cell suspension adjusted to g/ml 1.12 g/ml Culture medium Viable cells

38 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)

39 Publications database on cells OptiPrep  (since 1994) over 650 Nycodenz® (since 1984) approx 2000 Using either the Applications CD or the website: Follow the instructions to access the relevant Index Click on the cell of interest


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