843.766.5544 INFO@VITROLOGIC.COM

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

CELLULAR PRODUCTS

Don’t See What You Need? Let us know.