Complete Growth Medium
The base medium for this cell line is ATCC-formulated Dulbecco's Modified Eagle's Medium, Catalog No. 30-2002. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 15%
This medium is formulated for use with a 5% CO2 in air atmosphere. (Standard DMEM formulations contain 3.7 g/L sodium bicarbonate and a 10% CO2 in air atmosphere is then recommended).
To insure the highest level of viability, be sure to warm media and Trypsin / EDTA to 37ºC before using it on the cells. Cells should be split when they reach confluency. Split cells at approximately 0.4 X 104
- Remove and discard culture medium.
- Briefly rinse the cell layer with 1XPBS (SCRR-2201) solution to remove all traces of serum, which contain trypsin inhibitor.
- Add 5 mL of Trypsin-EDTA (0.25% (w/v) Trypsin-0.53 mM EDTA solution, ATCC# 30-2101) solution to flask and incubate for 1 minute, gently tapping the flask observe cells under an inverted microscope until cells detach (usually within 1 to 2 minutes).
- Add 6.0 to 8.0 mL of complete growth medium and rinse surface of the flask to detach all cells. Gently pipetting up and down will break cell clumps.
- Transfer all cells into a centrifuge bottle or tube and centrifuge at 271x g for 5 minutes.
- Remove and discard the supernatant
- Add 10 mL complete growth medium to cell pellet and with 10 mL pipette resuspend the cells gently (create a single-cell suspension).
- Add more complete growth medium to cell suspension as needed to plate cells.
- Place flasks in incubator @ 37°C with a 5% CO2 in air atmosphere.
Subcultivation Ratio: Plate the cells at approximately of 0.4 X 104 cells/cm2.
Medium Renewal: Twice a week or when pH decreases.
Liquid nitrogen vapor phase
Atmosphere: air, 95%; carbon dioxide (CO2), 5%