Materials required

1. 1X PBS Buffer
2. Histopaque 1077 medium
3. Freezing medium
4. Human blood
5. 20ml syringe with cannula
6. Heparinized green top tubes (4)
7. Centrifuge tubes(3)
8. Centrifuge
9. 1000 – 5000 microlitre procedure
10. Alcohol soaked cotton
11. 25ml glass pipette
12. 10ml glasspipette
13. Pipette pump
14. 50ml measuring cylinder
15. Waste beaker
16. 17.5ml cryovial (5)
17. Vial rack
18. Cryobox
19. -80 °C freezer
20. Liquid nitrogen freezer

Procedure

1. 20 ml of blood is collected from the patient’s vein.
2. The collected blood is transferred to four 7ml heparinized green top tubes (Total: 20ml).
3. Pool all the blood from the heparinized tubes were pooled together into one 50ml blue cap tube.
4. Made 1:1 dilution of whole blood with 1X PBS
5. Carefully underlay 10ml of histopaque-1077. 20ml of diluted blood is transferred to a 50ml tube.
6. Immediately centrifuge at 1800rpm [700g] for 30 min in a cooling centrifuge (4°C).
7. Carefully remove the tube from the centrifuge, observing the three layers: top is clear supernatant, middle is opaque fluid containing the PBMC, and bottom is RBC.
8. Carefully remove and discard the supernatant within 15 ml of the opaque layer of the PBMC. Quickly transfer the buffy coat layer of PBMC (around 5 ml) into a new 50ml tube.
9. Add 1x PBS to the PBMC suspension to obtain a final volume of 45 ml. Mix well and centrifuge at 1800rpm for 10 min in a cooling centrifuge (4°C).
10. Pour off and discard the supernatant and combine 1 x PBS to obtain the final volume of 30 ml. Mix well and centrifuge at 1500rpm (500g) for 10 min in a cooling centrifuge (4°C) to remove platelets.
11. Pour off and discard the supernatant. Resuspend the pellet in 5ml of the freezing medium (90%FBS+ 10%D MSO filter sterilized).
12. Aliquot 1ml of cell suspension per vial (cryogenic vial). Store at 80°C overnight. Next day transfer in the Liquid Nitrogen cryofreezer.