Cell Culture - Freezing, Thawing, and Adapting Cells

Biochemistry, Molecular Biology, and Cell Biology Protocols  >> Cell Culture - Freezing, Thawing, and Adapting Cells

Freezing cells

Whenever have a new cell line completed (e.g. after stable selection, or receiving a new cell line, or after adaptation into different media) freeze away a few vials of stocks
1. Grow cells to confluency on a plate or flask
2. For freezing media, use 20% fbs, 10% DMSO in DMEM media
3. Keep the freezing media on ice before resuspending cells
4. Pre-chill the tubes used for freezing
5. Put tubes with the cells in a slow-freezing container in the 80-degree freezer overnight
6. The next day, transfer to liquid nitrogen
7. Wear goggles or face protection when using the liquid nitrogen tank

Thawing cells

1. Thaw cells quickly in the 37-degree water bath to prevent them from dying (turn the tube in both directions in the water bath)
2. Pour it into a tube with regular media and centrifuge down cells
3. For stable cell lines, grow in regular media the first day, and change to selection media the next day

Adapting cells to new conditions

(e.g. suspension culture, new media)

1. Split cells at twice the concentration of its lower splitting dilution (perhaps even a 1:2 split)
2. When centrifuging cells, use only about 500x g for 5 minutes

To convert from g to rpm use this formula:
((g = 118 x 10-7 x r x n2))  
where g = relative centrifugal force
            r = rotating radius in cm
            n = rpm (revolutions per minute)

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