Thawing and Sorting Peripheral blood mononuclear cells (PBMCs)

A. THAWING PBMCs

1. Prepare media: RPMI with 10% FBS, 1x pen-strep, 1x Glutamine

2. 25 mL per sample with 1:10,000 benzonase (DNase)

3. Spin at 390g for 8 min in 10mL (use 15mL conical tube)

4. Aspirate and resuspend in 10mL

5. Filter through 70 micron strainer into 50 mL tube and transfer into 15mL tube

6. Spin 390g for 8 min

7. Resuspend and count cells

8. Rest for 45 – 60min @ 37 degrees in incubator at 1 – 2 million cells/mL

B. SORTING PBMCs

1. Spin down at 390g for 8 min

2. Wash with PBS one time
   

3. Use 2ul zombie per tube for 15 mins

4. Wash with sorting buffer (PBS with .1% BSA)

5. Blocking (5ul) and surface antibodies (treat each tube as 2 samples) for 30 mins

6. Wash 2x with sorting buffer

7. Pass through strainer

8. Read at 20 – 50 million cells/mL
   

1. RPMI with 10% FBS, 1x pen-strep, 1x Glutamine