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.
- Trypsinize cells as mentioned in the Subculturing Protocol (Steps 1-4).
- Transfer trypsinized cell suspension to a conical tube containing 40 ml of Growth Medium.
- Centrifuge at 1,000 rpm for 2-5 minutes at room temperature. Centrifugation at > 1500 rpm will result in cell death.
- Remove supernatant by gently pipetting it out without disturbing the cell pellet.
- Add the appropriate volume of Cryoprotectant Medium (DMEM high glucose 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 the isopropanol-filled cryo 1°C freezing container (be sure the tubes are capped tightly).
- Transfer the freezing container to -70°C overnight.
- The next day transfer frozen cells immediately to the liquid nitrogen vapor phase storage.
Note: After an overnight at -70°C, a vial of frozen cells should be tested to be sure that the frozen cells are still viable.