Complete Growth Medium
The base medium for this cell line is ATCC-formulated Eagle's Minimum Essential Medium, Catalog No. 30-2003. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%.
Volumes used in this protocol are for a 75 cm2 flask; 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 cold (less than 24°C) 0.25% (w/v) Trypsin - 0.03% 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 10 minutes).
Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach.
- Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
- Add appropriate aliquots of the cell suspension to new culture vessels.Cultures may be established with as few as 3 x 103 cells/mL.
- Incubate cultures at 22°C.
Medium Renewal: Once per week
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.
Temperature: 22°C; cells do not survive above 26°C but will grow at temperatures as low as 4°C.