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 500 ml of the base medium:
- fetal bovine serum (FBS) to a final concentration of 10%
- horse serum (HS) to a final concentration of 10%
- 0.1 mM non-essential amino acids
Volumes used in this protocol are for 75 cm2
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 Ca++/Mg++ free Dulbecco's phosphate-buffered saline (D-PBS) or 0.25% (w/v) Trypsin - 0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
- Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
- Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
- Transfer cell suspension to a centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes. Discard supernatant.
- Resuspend the cell pellet in fresh growth medium. Add appropriate aliquots of the cell suspension to new culture vessels. An inoculum of 2 x 104 to 4 x 104 viable cells/cm2 is recommended.
- Incubate cultures at 37°C.
Subcultivation ratio: A subcultivation ratio of 1:4 to 1:8 is recommended.
Medium renewal: Every 2 to 3 days
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.
Complete growth medium, 95%; DMSO, 5%. Cell culture tested DMSO is available as ATCC® Catalog No. 4-X.
Atmosphere: Air, 95%; carbon dioxide (CO2), 5%