Complete Growth Medium
Dulbecco's modified Eagle's medium with 4.5 g/L glucose and 0.1 mM non-essential amino acids; fetal bovine serum, 10%
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 0.25%Trypsin-0.53mM 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.
- Add appropriate aliquots of the cell suspension to new culture vessels
- Place culture vessels in incubators at 37°C.
Subcultivation Ratio: A subcultivation ratio of 1:3 is recommended
Medium Renewal: Twice 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.
Culture medium, 95%; DMSO, 5%. Cell culture tested DMSO is available as ATCC® Catalog No. 4-X.