I got this cell line from 2 different sources. It primarily belongs to ATCC and culture according to ATCC guideline. But in both cases cells die after 2nd passage. I am using 10% FBS in RPMI media.
NCH-1975, CRL-5908 can be maintained in culture medium, 95%; DMSO. Remove medium, and rinse the monolayer with fresh 0.25% trypsin, 0.53 mM EDTA solution. Remove the trypsin, add fresh trypsin and let the culture sit at room temperature (or at 37°C) until the cells detach (about 10 minutes). Add fresh medium, aspirate and dispense into new flasks. Sub culturing is done two –three times week, with a ratio of 1:3. Fetal bovine serum to a final concentration of 10% can be supplemented in RPMI-1640 Medium. There might be some problem with your FBS or FCS or do subculture some what earlier because so many autogenous factors also kill cells.
- Make sure the concentration of antibiotic very low not more than 100 micro gm streptomycine /ml and 100 IU of penicillin /mlin the media if you are using? the cells won't survive. Then we will see other factors.
What do the cells look like? Are they blebbing before death? Ruptured? Culturing with antibiotics can also mask (but not alleviate) contamination by mycobacteria. I never used antibiotic in my cultures. Perhaps you should try without and see if contamination shows up.
This article might be useful for differentiating between necrotic and apoptotic blebs.
http://www.nature.com/cdd/journal/v10/n6/full/4401236a.html
thank you so much for your kind advices. problem has been solved. i bought new cells and they are working pretty well. Thank you so much.
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