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. A split based on seeding density of 6 X 103 cells/cm2 is recommended.
Note: Volumes used in this protocol are for 75cm2 flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
Remove and discard culture medium.
- Briefly rinse the cell layer with 5.0 mL 1XPBS (ATCC Catalog No. SCRR-2201) solution to remove all traces of serum, which contain trypsin inhibitor.
- Add 3.0 mL 0.25% Trypsin-0.53 mM EDTA solution (ATCC Catalog No. 30-2101) solution to the flask and incubate for 2 minutes. Gently tap the flask and observe cells under an inverted microscope. Cells usually detach in 1 to 2 minutes.
- Add 3.0 mL complete growth medium and rinse the surface of the flask to detach all the cells. Gently pipette up and down will break cell clumps.
- Transfer all cell suspension into a centrifuge tube and centrifuge at 270 xg for 5 minutes.
- Remove and discard the supernatant.
- Add complete growth medium to the cell pellet and with 10 mL pipette gently resuspend the cells gently to create a single-cell suspension.
- Adjust volume as needed to seed vessels at approximately 6 X103 cells/cm2.
- Place flasks in incubator at 37° with 5% CO2 in air atmosphere.
Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 13 in Culture Of Animal Cells: A Manual Of Basic Technique by R. Ian Freshney, 5th edition, published by Wiley-Liss, N.Y., 2005.
Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:7 is recommended
Medium Renewal: Twice a week or when pH decreases
Complete growth medium supplemented with an additional 40% FBS and 10% (v/v) DMSO
Cell culture tested DMSO is available as ATCC Catalog No. 4-X.
Atmosphere: Air, 95%; carbon dioxide (CO2), 5%