- 20ml syringe with cannula
- Heparinized green top tubes (4)
- 1000 – 5000 microlitre procedure
- 20 ml of blood is collected from the patient’s vein.
- The collected blood is transferred to four 7ml heparinized green top tubes (Total: 20ml).
- Pool all the blood from the heparinized tubes were pooled together into one 50ml blue cap tube.
- Made 1:1 dilution of whole blood with 1X PBS
- Carefully underlay 10ml of histopaque-1077. 20ml of diluted blood is transferred to a 50ml tube.
- Immediately centrifuge at 1800rpm [700g] for 30 min in a cooling centrifuge (4°C).
- 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.
- 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.
- 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).
- 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.
- Pour off and discard the supernatant. Resuspend the pellet in 5ml of the freezing medium (90%FBS+ 10%D MSO filter sterilized).
- Aliquot 1ml of cell suspension per vial (cryogenic vial). Store at 80°C overnight. Next day transfer in the Liquid Nitrogen cryofreezer.