LYMPHOCYTE
SEPARATION from WHOLE BLOOD
Protocol:
Dispense 3ml of well-mixed
Ficoll-Hypaque 1.077 (Sigma) into a 15ml conical centrifuge tube.
Dilute 2ml whole blood (18-20° C)
with an equal volume of sterile balanced salt solution (PBS). Note: use fresh
venous whole blood anticoagulated with EDTA or heparin (not citric acid).
Layer the diluted blood onto the
surface of the Ficoll-Hypaque. Do not allow the two solutions to mix.
Centrifuge at 400xg for 30-40 minutes at 18-20° C.
Draw off the upper plasma layer
using a clean, sterile Pasteur pipet, leaving the lymphocyte layer at the
interface undisturbed (this layer is referred to as the buffy coat).
Using a clean, sterile Pasteur
pipet, aspirate the buffy coat in a minimum volume (transfer to a fresh 15ml
conical centrifuge tube). Wash the cells two times in at least three volumes of
sterile PBS (pellet the cells by centrifugation at 1100rpm for 10 minutes).
Resuspend the cells in complete
RPMI 1640
Ref:
Boyum, A. (1968). Isolation of
mononuclear cells and granulocytes from human blood…
Liddell and Cryer (1991). A
Practical Guide to Monoclonal Antibodies. John Wiley and Sons, Ltd. p. 81.