Sep 07, 2021
PBMC Thawing Protocol
- Fang Zhang1
- 1Fang Zhang
- czi
Protocol Citation: Fang Zhang 2021. PBMC Thawing Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bm9ck92w
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: October 10, 2020
Last Modified: September 07, 2021
Protocol Integer ID: 43012
Keywords: PBMC, thawing, cell culture, RPMI,
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
Materials
Reagents:
- PBMC washing medium
• A: RPMI-1640 with 5 to 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine
• B: 50% X-vivo 15 medium (Lonza) + 25U/ benzonase
• C: 1X CTL-Anti-Aggregate-Wash™ (CTL)
- PBMC complete culture medium
• RPMI-1640 with 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine
Materials:
- 50 mL falcon tube (Fisher scientific #14-432-22)
- Trypan blue
- Water bath
- Dry ice
- 70% EtOH
Safety warnings
Please refer to Safety Data Sheets (SDS) for health and environmental hazards.
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 25 mL warmed washing buffer .
Use 1 mL washing medium to rinse out the cryovial and gently mix the cells by inverting the 50 mL Falcon tube ~5x.
Wash 1:
Spin the cells: 400 x g, Room temperature, 00:08:00 . Pour off the supernatant.
Wash 2:
Suspend the cell pellet in 1 mL prewarmed medium (dropped slowly along the side of the tube) and resuspend the cell pellets, add 9 mL complete medium . Spin the cells: 400 x g, Room temperature, 00:08:00 .
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.