Cell Culture Information:
Cells become confluent at a density of approximately 40,000 cells/cm^2, and a saturation density of about 50,000 cells can be reached. However, in culture, cells should not be allowed to become completely confluent. Cells will double every 18 hours.
Culture cells in a plastic flask (they do not adhere well to certain types of glass). Use DMEM formulated with 10% FBS.
- Remove medium and rinse with Trypsin-EDTA (0.25% Trypsin, and 0.53 mM EDTA)
- Add an additional 1-2 mL of Trypsin-EDTA and allow flask to sit at room temperature until cells begin to disperse (round up). Dispersal can be encouraged by incubating.
- Centrifuge and removed Trypsin-EDTA supernatant.
- Add fresh culture medium to cells and aspirate.
- Dispense suspended cells into new culture flasks at a density of 3 x 10^5 cells per plate or 4 x 10^5 cells per flask.
Subculture ever 3 days. Renew medium 2 times a week.
Freeze cells in conditioned growth medium supplemented with 5% (v/v) DMSO and store in the liquid nitrogen vapor phase or at -135 °C.