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