Hi,
I want to induced hypoxia in cancer cell lines to check regulation of VEGF signaling components.
So is there any simple way to induced hypoxia in breast cancer cell lines like HCT116, MCF7 or T47D (which we have) by some kinds of chemicals or another way like to reduced oxygen supply in incubator?
The answer is Yes. However, depending upon the goal, both chemically-induced hypoxia and a reduction in the oxygen levels in a cell culture incubator may be appropriate. Further, each cell line may require some optimization with respect to concentrations of chemicals (either cobalt chloride or desferoxamine) and the time of incubation to induce hypoxia that results in appreciable increases in HIF-1alpha levels. If possible, a reduction in oxygen levels is a preferred method to reproducibly induce hypoxia and acute hypoxia in cells in culture.
there are many ways..one can be to use a special incubator with 1% hypoxia or to use the PHD inhibitor DMOG in normoxic condition. You can also use conbalt cloride CoCl2
If you have no access to a hypoxia incubator, there is a relatively easy alternative available. You can place the cell culture dishes in a bag which also contains an oxygen absorbing chemical (basically ascorbic acid, I think). Oxygen is replaced by carbon dioxide, which might also shift the pH to slightly more acidic conditions, such as in ischemia. Such devices can be bought as "anaerob atmosphere generation bags", Anaerocult or similar. YOu simply put your dishes in a tight plastic bag, which is equipped with a bag filled with the wetted oxygen absorber. The depeltion of oxygen can be followed by an indicator (Anaerotest), if you like. We have compared these bags with our hypoxia incubator and find more or less the same response in MCF-7 cells (HIF accumulation and VEGF-secretion).
Hypoxia chamber allows an adjustment of a range of oxygen levels:
3% : close to levels in human issues/organs
2-1%: hypoxia (seen in tumors)
0.1%: an acute hypoxia
Depending upon cell types, responses vary very significantly to the above three levels of oxygen levels.
For MCF7, T-47D ... CoCl2 100 micro molar 6 Hours. Antibody for detection Anti- human HIF1a BD ( Cat# 610959) dilution 1/200. Good luck.
Guys I corrected the concentration of CoCl2 6 hours. The stock solution is 25 mM and concentration to induce Hif1a is 100 micro molar 6hs. Sorry for the mistake
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