CHO cells grow in F-12 media supplemented with 10% FBS.

Thaw cells in 37oC water bath.

Put the thawed cells into F-12 media[1],[2].  It may take several days for the cells to become confluent. (Cells expressing IRS-1 or any IRS-1 mutants should be maintained in histidinol, cells expressing the insulin receptor are neomycin resistent, but because these cell lines are very stable it is not necessary to maintain them in drug-containing media).


Splitting confluent cells

Aspirate off the media from a 75cm2 flask of cells.

Wash with 5 ml PBS-CMF (sterile).  Aspirate off the PBS.

Add 3 ml trypsin (low trypsin stock found at -20oC) and let incubate on cells until cells round up and detach when flask is tapped lightly.  (Do not let trypsin sit too long or the receptor will be cleaved.)

To stop trypsin reaction add 9mls F-12 + FBS (10%).  To maintain cells, a 1:10 dilution is good.

Resuspend cells and add 1 ml to new flask which contains 14mls media.


Freeze cells

Freeze cells by spinning down cells after media has been added to stop trypsin reaction.

Resuspend cells in F-12 + 20% FBS + 10% DMSO and aliquot to cryovials (1ml/vial).

Freeze at -20oC for .1 hour, -70oC overnight and transfer to liquid nitrogen tank.

1[1]For histidinol resistance:  Dilute stock solution 1:10 in F-12 media.

2[2]Add 10 ml media to 0.8g geneticin, dissolve geneticin.  Filter sterilize media + geneticin mixture, with a syringe, directly to 1L bottle of media. (All overexpressing insulin receptor cells)