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cho-k1 culture - (Sep/08/2005 )

hi,everyone , i am a newcomer to cell culture.The first kind cell I handled is the cho-k1.
But the cell's growth is not good.After each thawing, the cell can grow no more than 3 days,then the growth condition of the cell will be bad, there are a lot of dark dots in the cell and the cell grows very slow,the culture medium is F12 with 12% FBS and biantibitics.
I need your sugestions!
thank you


atcc web site recommends as follows : (extract from this webpage

Propagation: ATCC complete growth medium: Ham's F12K medium with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate., 90%; fetal bovine serum, 10%

Temperature: 37.0 C

Subculturing: Protocol:

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
3. 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.
4. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
5. Add appropriate aliquots of the cell suspension to new culture vessels.
6. Incubate cultures at 37�C.