PBMC Thawing Protocol
Fang Zhang
Abstract
This protocol details methods for thawing peripheral blood mononuclear cells (PBMC).
For a protocol detailing Culture and Stimulation, please view the following: PBMCs Culture and Stimulation.
For a protocol detailing Cell Staining for Flow Cytometry Assay, please view the following: Cell Staining for Flow Cytometry Assay.
Attachments
Steps
Warm Washing buffer and medium to 37°C in a water bath.
Remove vials from liquid nitrogen and transport them to the lab on dry ice.
Thaw frozen vials, only 1 vial at a time, in a 37°C water bath. When cells are nearly completely thawed, carry the vials to the hood and swab them with 70% EtOH.
Gently remove PBMCs (avoid pipetting up and down, as the cells are very fragile at this stage) and transfer the cells into a 50 mL falcon tube (Fisher scientific #14-432-22) containing 25mL.
Use 1mL to rinse out the cryovial and gently mix the cells by inverting the 50 mL Falcon tube ~5x.
Wash 1:
Spin the cells: 400x g. Pour off the supernatant.
Wash 2:
Suspend the cell pellet in 1mL (dropped slowly along the side of the tube) and resuspend the cell pellets, add 9mL. Spin the cells: 400x g.
If cells were thawed in the presence of benzonase, perform an additional wash with culture medium in the absence of benzonase.
Count cells and determine viability by Trypan blue staining.
