When we prepare the medium for CRL-2254 we use 5 mL of ITS solution (Sigma I-1884 dissolved in 2% glacial acetic acid in sterile cell culture grade water), 1 mL of Dexamethasone (Sigma D-8893), 50 mL of FBS, and 444 mL ATCC DMEM/F-12 medium (30-2006). Dexamethasone is a steroid analog (glucocorticoid) which stimulates glucose supply to the cells so it is important. To make the dexamethasone we filter sterilize 25 mL of 200 proof ethanol. Then dissolve 1mg of dexamethasone Sigma D8893 in 1mL of absolute ethanol. Add 49 mL of sterile serum-free medium and mix with a sterile stir bar for 10 minutes or until dissolved then aliquot the solution into sterile tubes for use in the complete medium.
It is normal for cultures of CRL-2254 to have many cells remaining in suspension for the first few days after thaw. These viable floating cells should be retained by gentle centrifugation and adding back to the original culture when feeding with fresh medium every 2 or 3 days. It is important to keep these floating cells in the same culture vessel as the adherent population. If they are separated (or discarded), the culture may become too dilute which can cause growth to slow or stop.
|Date Created||12/18/2017 10:37 AM
|Date Updated||12/18/2017 10:37 AM