Start with everything at room temperature:

  Add 15 mL Ficoll-Paque Plus to each SepMate tube by pipetting directly into centre of plastic separator (try not to introduce a lot of air beneath the separator)

  Pool anti-coagulated blood tubes (3 x 9 mL Li Hep tubes) – should come to 27ml

  Dilute 1:1 in PBS + 2% FBS (now have ~54 mL diluted blood)

  Layer blood on top of Ficoll-Paque Plus by gently pipetting down the side of the SepMate tube (avoid pipetting directly above the small notches as this lets blood through to below the separator)

  Cap tightly and spin at 1200 x g, 10m, RT, accelerator and break on full 

  Cool PBS + 2% FBS in refrigerator and set centrifuge to cool (4C, though 10C should be sufficient)

  Pour interface off SepMate tube into new 50 mL falcon (2 SepMate tubes per falcon – if odd number of tubes, even out the volumes between falcons)

  Top cells up to 45 mL with chilled PBS + 2% FBS, mix well

  Centrifuge 900 x g, 10m in cooled centrifuge

  Meanwhile, centrifuge 5 mL serum tube in the same spin

  Discard supernatant and flick to dislodge pellet

  Resuspend cells in chilled PBS + 2% FBS and pool cells from each individual into 1 tube with 40 mL buffer

  Centrifuge 300 x g, 10m in cooled centrifuge

  Meanwhile, aliquot serum (avoid any red) and store aliquots in freezer (these do not contain any human cells)

  Discard supernatant and flick to dislodge pellet

  Resuspend cells in 25 mL chilled PBS + 2% FBS

  Take 10 uL aliquot and count on haemocytometer

  Centrifuge 300 x g, 10m in cooled centrifuge

  Discard supernatant and pipet off final drips, flick to dislodge pellet  

  Resuspend cells in chilled 1-1.5 mL FBS + 10% DMSO (if more than 15 million cells, make multiple aliquots) – Freeze in ~5million cell aliquots

  Rapidly move cryovials to Mr Frosty and put in -80C Freezer