Preparation of Frozen Stock (C2009)
Culture should be approximately 90% confluent prior to preparation of frozen stock in order to insure the highest number of viable cells from reviving a frozen culture.
- Transfer one-day old growing culture into a sterile 50 ml conical tube.
- Centrifuge culture at ≤ 1,500 rpm for 2-3 minutes at room temperature. Centrifugation at >1500 rpm will result in cell death.
- Remove and discard supernatant by gently pipetting it out without disturbing the cell pellet.
- Resuspend cell pellet in 10 ml of RPMI-1640 with 10% FBS. Repeat centrifugation and remove and discard supernatant by gently pipetting it out without disturbing the cell pellet.
- Add the appropriate volume of Cryoprotectant Medium (RPMI-1640 containing 50% FBS and 10% DMSO) to the cell pellet to achieve a concentration of at least 2 x 106 cells per ml.
- Resuspend pellet by gently pipetting up and down 2-3 times.
- Aliquot 0.5-1 ml of cell suspension into cryogenic tubes.
- Place tubes in an isopropanol-filled cryostorage container (be sure the tubes are capped tightly).
- Transfer the cryostorage container to -70°C overnight.
- The next day transfer frozen cells immediately to liquid nitrogen vapor phase storage.
Note: After an overnight at -70°C, a vial of frozen cells should be revived and tested to ensure that the frozen cells are still viable and functional.