I have collected 10 ml blood in heparinized vacutainers. The pellet seems smaller than when I collect in EDTA ones.
I cryopreserve in 10% DMSO, 40% human serum and 50% rpmi 1640 and then freeze slowly.
What can I do to optimise yield as I shall be checking for NK cell functional subtypes as well as perforin and granzyme B?
I do not work with NK cells, however T cells are in a preactivated state when you freeze PBMCs and perform assays with T cells after thawing.
Thanks, Dr Crauwels. I was concerned that I should get enough viable NK cells after thawing.
Hi Anjali, in our lab we follow a freezing protocol according to Immune monitoring standards.
Solution A: 60% AB serum+40% RPMI
Solution B: 80% Serum+20% DMSO
PBMCs are resuspended in 1ml volume (0.5ml solutionA+0.5ml solution B) and frozen gradually overnight in -80 deg C and the next day stored in Liquid N2.This protocol works well and has a higher recovery after thawing. Works well for all cell types (T cells, B cells, NK, etc) and used for immune monitoring purposes.
Sometimes while thawing a vial, it is better to add benzonase, as it prevents cell clogging and better for use like FACS/functional assays, etc. Hope it helps. All the best!
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