Procedure
A. THAWING PBMCs
Prepare media: RPMI with 10% FBS, 1x pen-strep, 1x Glutamine
25 mL per sample with 1:10,000 benzonase (DNase)
Spin at 390g for 8 min in 10mL (use 15mL conical tube)
Aspirate and resuspend in 10mL
Filter through 70 micron strainer into 50 mL tube and transfer into 15mL tube
Spin 390g for 8 min
Resuspend and count cells
Rest for 45 – 60min @ 37 degrees in incubator at 1 – 2 million cells/mL
B. SORTING PBMCs
Spin down at 390g for 8 min
Wash with PBS one time
Use 2ul zombie per tube for 15 mins
Wash with sorting buffer (PBS with .1% BSA)
Blocking (5ul) and surface antibodies (treat each tube as 2 samples) for 30 mins
Wash 2x with sorting buffer
Pass through strainer
Read at 20 – 50 million cells/mL
Recipes
RPMI with 10% FBS, 1x pen-strep, 1x Glutamine