Isolation of Mononuclear Cells

HISTOPAQUE 1077:

(1) Add appropriate amount (1:1 ratio with volume of blood) of cold HISTOPAQUE 1077 into tube. (ex. you have 8 ml blood, one way is to add 4ml to 2 15 ml tubes)

(2) Carefully layer blood onto HISTOPAQUE.  (dispense it slowly down walls near surface) (ex. add 4 ml blood to each of the tubes (1).

(3) Centrifuge at 340xg (10,000 RPM) for exactly 30 minutes at room temp (centrifuging at lower temps may cause clumping) (centrifuge is outside Rm 4022; set at 28 on black dial)(do not apply brakes to stop)

(4) Carefully transfer (using bulb pipette) plasma top layer to within 0.5 cm of the opaque interface containing the PBMCs and discard.

(5) Carefully transfer the opaque interface with a bulb pipet into clean tube (dispense it down the side of this tube) (eg., use 4 new tubes in this example)

Washing

(6) add HBSS to tube from refrigerator

(7) centrifuge for 10 min at 300 x g (950 RMP).

(8) Aspirate the supernatent and discard and resuspend the pellet with HBSS. Centrifuge as in (7)

(9) repeat washing step again as in 8

(10) prepare medium (10% serum + RPMI + PSG (glutamine+Strep+Penecillin) (these are the glass tubes in freezer part of the refrigerator). Pour off supernatent in (11) and resuspend the pellet in say 5 ml of medium and count cells for experiment.