Freezing Cells

Materials:

    • Cell culture media

    • PBS

    • Trypsin

    • FBS

    • Sterile-filtered DMSO

    • Cyrovials

    • Freezing media – 45% media + 50% FBS + 5% DMSO

Preparation:

    • Warm media, trypsin, PBS, and FBS in water bath

    • Wipe laminar flow hood down with 70% ethanol

    • Wipe all containers that go into hood with 70% ethanol

Procedure:

    1. Aspirate old media from dishes

    2. Add 8 ml PBS

    3. Aspirate off PBS

    4. Add 4 ml trypsin

    5. Place dish in incubator for 2-6 minutes

    6. Remove dish from incubator and gently tap to loosen cells. Make sure all cells have lifted off dish – if not put back in incubator for 1-2 more minutes.

    7. Add 4 ml media to dish

    8. Remove all liquid of dish to 15 ml centrifuge tube

    9. Rinse dish with PBS and move to centrifuge tube

    10. Centrifuge at 1000 rpm for 5 min.

    11. Aspirate off as much supernatant as possible.

    12. Resuspend cells in 1 ml media.

    13. Perform cell count (Coulter counter) to determine number of cryovials needed (1-2 million cells/cryovial)

    14. Add appropriate amount of media and FBS to cell suspension. Add the DMSO last as it is toxic to cells.

    15. Add 1 ml of cell suspension to each cryovial.

    16. Place cryovials in -80 ºC freezer