Human peripheral blood mononuclear cells (PBMCs) are frequently used in the study of biological processes and drug development, and for applications such as in vitro cell-based assays. This protocol covers how to thaw cryopreserved PBMCs using RPMI medium.
- Vial of cryopreserved PBMCs
- 37°C Water Bath
- RPMI with L-Glutamine and 10% FBS
- 15mL tubes
- Pipets and pipet dispenser
- CO2 incubator
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.