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  • Protocol for Preparation of Frozen Stock

    Introduction

    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.

    Protocol

    1. Trypsinize cells as mentioned in the Subculturing Protocol (Steps 1-4).
    2. Transfer trypsinized cell suspension to a conical tube containing 40 ml of Growth Medium.
    3. Centrifuge at 1,000 rpm for 2-5 minutes at room temperature. Centrifugation at > 1500 rpm will result in cell death.
    4. Remove supernatant by gently pipetting it out without disturbing the cell pellet.
    5. 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.
    6. Resuspend pellet by gently pipetting up and down 2-3 times.
    7. Aliquot 0.5-1 ml of cell suspension into cryogenic tubes.
    8. Place tubes in the isopropanol-filled cryo 1°C freezing container (be sure the tubes are capped tightly).
    9. Transfer the freezing container to -70°C overnight.
    10. 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.