FICOLL-HYPAQUE SEPARATION OF HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC) FROM WHOLE BLOOD
MATERIALS
- Whole blood unit or vacutainer tubes (with anti-coagulant)
- Dulbecco’s Phosphate Buffered Saline (DPBS), sterile
- Ficoll-Hypaque separation media
- Fetal Bovine Serum, heat-inactivated
- DMSO, sterile for freezing
- Trypan Blue Stain, 0.4%
EQUIPMENT
- Centrifuge, swinging bucket
- Biosafety cabinet
- Micropipettors (200µL and 1000µL)
- Pipets, sterile, individually wrapped 10mL and 25mL
- Centrifuge tubes, sterile, 50mL
- Pipetman
METHODS
Collect Plasma
- Centrifuge whole blood unit or vacutainer tubes at 1500-1800rpm for 10-12 minutes
- Remove plasma layer and store for future use (aliquot and freeze at -80°C)
Separate PBMC
- Separation of PBMCs is performed at room temperature
- Dilute whole blood 1:2 with DPBS in a sterile container (if plasma removed, replace plasma volume with DPBS prior to diluting)
- Prepare 50mL centrifuge tubes by adding 11-13mL of Ficoll-Hypaque to each tube
- Overlay the 50mL tubes prepared above with approximately 35-40mL of diluted whole blood
- Centrifuge for 25 minutes at 1250rpm with no brake
- Remove large portion of diluted plasma layer being careful not to remove the interface of mononuclear cells
- Remove interface layer taking a large portion of the Ficoll-Hypaque layer down to the RBC pellet and put into new 50mL tube
- Add a minimum of 2X the volume of interface removed or fill 50mL centrifuge tube with DPBS to wash
- Centrifuge at 1250rpm for 10 minutes
- Pour off or aspirate supernatant and flick tube to resuspend PBMC pellet
- Fill tube to 50mL with DPBS for 2nd wash and centrifuge for 5 minutes at 1250rpm
- Repeat 1 additional time for a total of 3 washes
- Following final wash, resuspend cells in culture medium
- Count cells by Trypan Blue Exclusion Method or automated cell counter
- Adjust concentration of cells in growth medium or freeze aliquots for future use