Each time a new cell line has been completed (eg, stable selection, or receive a new cell line, or after adaptation in different media) freeze away several bottles of stock
- Growing the cells to confluency in plates or flasks
- For the freezing media, the use of 20% FBS, 10% DMSO DMEM media
- Keep frozen media on the ice before resuspending cells
- Pre-chilled tubes used for freezing
- Put the tube with the cells in containers slow freezing in the freezer overnight to 80 degrees
- The next day, transfer to liquid nitrogen
- Wear goggles or face protection when using liquid nitrogen tank
thawing cells
- Thaw the cells quickly in a water bath of 37 degrees to prevent them from dying (change tubes in both directions in a water bath)
- Pour into a tube with regular media and centrifuge bottom of the cell
- For the stable cell lines, growing in regular media first day, and a change to the selection media the next day
Cells adapt to the new conditions
(For example, suspension culture, new media)
- Split the cell divides twice the concentration of a lower dilution (possibly even 1: 2 split)
- When centrifuging the cell, use only about 500x g for 5 minutes
To convert from g to rpm using 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)