How to Thaw PBMCs

Note: follow all safety precautions, and ensure you have the required equipment, materials and reagents to perform the following protocol.

1. Warm water bath to 37℃, and ensure RPMI is warmed to 37℃.
2. 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.
3. Place cryopreserved cell vial in 37℃ water bath. Submerge cryovial halfway and thaw for approximately 2 minutes. Gently swirl vial.
4. Examine vial, continue 37℃ thaw until ice just before last ice crystal has melted. Do not allow vial to warm to greater than 10℃.
5. Transfer vial to a biosafety cabinet and wipe the outside of the vial with 70% ethanol or isopropanol.
6. 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.
7. Rinse cryovial with 2 mL pre-warmed RPM using a pipet. Pour cells into 15 mL conical tube.
8. Incubate cells for 5 minutes at 37℃.
9. Centrifuge cell suspension at 260 x g at 20℃ (room temp) for 5 min, low brake.
10. Pour off the supernatant.
11. Add 2 mL pre-warmed RPMI using a pipet. Mix by flicking tube (avoid pipetting).
12. Incubate cell suspension for 1 hour to overnight in a 37℃ CO2 incubator. Leave cap loose so gas transfer can occur.
13. Cells are now ready for use in downstream applications.