RAT-1 cells grow in F-12/DMEM (1:1) media supplemented with 10% FBS.


Thaw cells in 37oC water bath.  Put the thawed cells into media.  It will take several days for cells to become confluent.


SPLITTING CONFLUENT CELLS (when maintaining a 75cm2 flask of cells):


1.  Aspirate off the media.

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

3.  Add 1 ml trypsin (0.1%) and let incubate on cells until cells round up and detach when

     flask is tapped lightly.  (Do not let trypsin sit too long or receptor will be cleaved.)

4.  To stop trypsin reaction add 9ml media. 

5.  Resuspend cells and remove desired volume to new flask which contains media.





Freeze cells by spinning down cells after media has been added to stop trypsin reaction.  Resuspend cells in media + 10% DMSO and aliquot to cryovials.  Freeze at -20oC for 1 hour, -70oC overnight and transfer to liquid nitrogen tank.