Hello....
I am intending to mimic hypoxia induced conditions in cancer cells with CoCl2 (for 24 hrs) and then re-oxygenate the CoCl2 treated cells with fresh culture media without CoCl2.
I am confused to decide the optimum cell confluence at which i should add CoCl2 to my cells and after 24hr post CoCl2 treatment, how long should I run the cells in fresh media?
if I treat the cells with CoCl2 at 60% confluence, the same cells may reach about 80-95% confluence in 24 hrs and is it wise to replenish the 80-90% confluent cells with fresh media and grow for next 12 or 24 hrs?
Any suggestion would of immense help...
Thanking you in advance
Thanks Sandra Bernaldo de Quiro's,
Actually, I don't have to treat my cells with any kind of drug at this level. My aim is to see the effect of hypoxia (HIF 1 Aplha) on cellular metabolism and metastasis. I have to do western blot for some proteins....
I will be evaluating both hypoxia mediated changes in certain protein expression levels as well as the effect of Reoxygenation (of hypoxia treated CoCl2 treated cells) on the same proteins....
I am not sure as to which confluence should I treat with CoCl2 and after 24 Hrs post CoCl2 treatment, again I am supposed to reoxygenate my CoCl2 treated cells with normal media (without CoCl2) to reoxygenate them for about 16- 24 hrs.
Like today I harvested one set of experiment, in which I treated my cells with CoCl2 for 24 hrs and the confluence at treatment time was 60%. After incubating the cells for 24 hrs, i changed Cocl2 containing media with fresh media and the confluence was about 80-85% at that time. Reoxygenation was continued for 16 hrs and today when I observed the cells , they were almost fully confluent.
Won't the cell confluence affect the protein expression levels or it will be okay to go for WB to assess the expression leves?
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