In this video, we will demonstrate how to thaw peripheral blood mononuclear cells,or PBMCs, for maximum cell viability and recovery.
Human PBMCs are frequently used in the study of biological processes and drug development, and for applications such as in vitro cell-based assays.
Fresh PBMCs are often cryopreserved and stored in liquid nitrogen if they are not needed immediately for research applications. Researchers with limited access to donors or ability to isolate immune cells from whole blood may purchase ready-to-use, cryopreserved PBMCs from biospecimen suppliers like Cytologics.
Proper handling and thawing of these cells is critical for obtaining optimal viability and recovery. This instructional video covers best practices for thawing PBMCs prior to use in downstream applications.
In addition, we’ve included our protocol to thaw PBMCs protocol using RPMI medium below(PDF version available for download at the bottom of the page).As thawing protocols for specific cell types may vary, always refer to the recommended protocol received with your cells when ordering from Cytologics.
Key Reagents and Supplies
- RPMI with L-Glutamine and 10% FBS
- 15 mL tubes
- Pipets and pipet dispenser
- 37℃ water bath
- CO2 incubator
Steps for Thawing PBMCs
Note: follow all safety precautions, and ensure you have the required equipment, materials and reagents to perform the following protocol.
- Warm water bath to 37℃, and ensure RPMI is warmed to 37℃.
- When removing frozen cells from liquid nitrogen storage, it is important to minimize exposure to room temperature (15-25°C). If not proceeding directly to thawing, place the cells on dry ice or in a liquid nitrogen container.
- Place cryopreserved cell vial in 37℃ water bath. Submerge cryovial halfway and thaw for approximately 2 minutes. Gently swirl vial.
- Examine vial, continue 37℃ thaw until ice just before last ice crystal has melted. Do not allow vial to warm to greater than 10℃.
- Transfer vial to a biosafety cabinet and wipe the outside of the vial with 70% ethanol or isopropanol.
- Pour thawed cells into 15 mL conical tube containing 5 mL of RPMI that has been pre-warmed to 37℃. Do not use a pipet for this step.
- Rinse cryovial with 2 mL pre-warmed RPM using a pipet. Pour cells into 15 mL conical tube.
- Incubate cells for 5 minutes at 37℃.
- Centrifuge cell suspension at 260 x g at 20℃ (room temp) for 5 min, low brake.
- Pour off the supernatant.
- Add 2 mL pre-warmed RPMI using a pipet. Mix by flicking tube (avoid pipetting).
- Incubate cell suspension for 1 hour to overnight in a 37℃ CO2 incubator. Leave cap loose so gas transfer can occur.
- Cells are now ready for use in downstream applications.
Also, click the link below to download a free protocol for thawing PBMCs as demonstrated in the video to keep with you in the lab!