Materials required

• 1X PBS Buffer

• Histopaque 1077 medium

• Freezing medium

• Human blood

• 20ml syringe with cannula

• Heparinized green top tubes (4)

• Centrifuge tubes(3)

• Centrifuge

• 1000 – 5000 microlitre procedure

• Alcohol soaked cotton

• 25ml glass pipette

• 10ml glasspipette

• Pipette pump

• 50ml measuring cylinder

• Waste beaker

• 1.5ml cryovial (5) 

• Vial rack

• Cryobox

• -80 °C freezer

• Liquid nitrogen freezer

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.