We are attempting to induce hypoxic reponse in various breast cancer cells lines with the use of CoCl2 (more specifically using stock of 0.1M CoCl2 that was ordered already prepared and adding various amounts directly to cell media (i.e. - serum free media) for a final concentration from 50um-300um ) and for various amounts of time 6 hours-72 hours. However, instead of seeing an expected increase in HIF alpha both bia qPCR and Western Blot (cytoplasmic and nuclear fractions) following treatment with CoCl2, we are unsure why we are seeing decreased or unchanged HIF alpha levels.
i have a protocol which works fine for me in hek 293 as well as colon cancer cell lines however we don't use serum free media we use with media with FBS as routine.
first make 200um cocl2 stock.
to 10ml of fresh media+FBS we add 5ul of cocl2 from stock add to the cells
then we keep cells incubation for different time points like 12hr, 24hr, 48hr,72hr
i see maximum hif induction at 48hr incubation post 48hr i dont see any significant difference. hope this works for you
good luck
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