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:
Aspirate old media from dishes
Add 8 ml PBS
Aspirate off PBS
Add 4 ml trypsin
Place dish in incubator for 2-6 minutes
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.
Add 4 ml media to dish
Remove all liquid of dish to 15 ml centrifuge tube
Rinse dish with PBS and move to centrifuge tube
Centrifuge at 1000 rpm for 5 min.
Aspirate off as much supernatant as possible.
Resuspend cells in 1 ml media.
Perform cell count (Coulter counter) to determine number of cryovials needed (1-2 million cells/cryovial)
Add appropriate amount of media and FBS to cell suspension. Add the DMSO last as it is toxic to cells.
Add 1 ml of cell suspension to each cryovial.
Place cryovials in -80 ºC freezer