Protocols
PBMC Isolation Protocol
- Freshly isolated human whole blood (20mL/tube) was layered over Sigma Histopaque®-1077 (20mL/tube) in 50mL conical tubes at room temperature.
- Samples were centrifuged at 400xg in a swinging bucket clinical centrifuge for 30 minutes at room temperature with no brake.
- The white cellular layer at the interface between the plasma and density gradient was removed by pipet.
- RPMI (serum free) was added at 1:1 volume. These peripheral blood mononuclear cells (PBMC) were spun down at 250xg for 10 minutes.
- A 1:10 dilution of red cell lysis buffer (Biolegend) was added for 10 mins then washed with 35mL RPMI.
- PBMC’s were washed 1 time with RPMI-1640 (serum free) by dilution and centrifugation for 10 minutes at 250xg.
- The washed PBMC were resuspended in RPMI-1640 + 10% FBS and plated at 1x106 cells/mL.
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