Isolation of PBMCs From Whole Blood

Collect 36mL whole blood into EDTA tubes

Centrifuge the blood samples at 1000x g,25Room temperature

Carefully remove collection tubes from centrifuge; plasma is the top layer above the rest of the whole blood layer

Using caution not bring up the remaining whole blood layer, remove plasma and aliquot it into 1.5mL microcentrifuge tubes

Centrifuge the isolated plasma at 1600x g, to remove residual cells and debris

After centrifugation, aliquot the plasma supernatant into 1mL aliquots by using microcentrifuge tubes labeled with patient ID

Freeze at -80°C.

Dilute the rest of the whole blood sample to a 1:1 volume ratio with the PBS

Add 15mL density gradient medium to a fresh 50mL conical tube and gently layer the diluted blood on top of the density gradient medium. Take care not to mix the two layers.

Centrifuge at 800x g

Carefully harvest the cells by inserting the pipette directly through the upper plasma layer to the mononuclear cells at the interface. Alternatively, you can first remove the upper layer and then collect the cells

Wash the harvested PBMC in 12mL of PBS

Centrifuge at 300x g

Discard the surnatant and wash the PBMC pellet in 5mL of PBS

Centrifuge at 300x g

Discard the surnatant and resuspend the cell pellet in 5mL of PBS

Count the cells using Trypan blue staining.

Resuspend PBMCs to 5x10⁶ cells/mL in PBS and aliquot them in cryotube vials (1mL/vial) labeled with patient ID

Centrifuge at 800x g

Discard the surnatant and store the cell pellets at -80°C