When I expose my primary cortical neurons to hypoxia(1%) in a professional hypoxic workstation, only about 10% of the cells die at 24 hrs. Even at 48 hrs, it just reaches up to 20%-30%. Is it normal? Should it be that neurons are more susceptible to hypoxia? Thanks~
Did you add any protein synthesis inhibitors in the culture media? Survival signaling could be activated under this condition.
you have to consider about your exposure time, for hypoxic condition and the standardize the protocol so on , so forth, to achieve the percentage u want for experimental work
You may try to add cycloheximide to inhibit protein synthesis. Then you could try to induce apoptosis. Hope it helps.
I induced apoptosis in my cell lines with TNF-a for 30 min after 4 hours of cycloheximide pre-incubation in starve media.
Standard cell cultures exposed to 1% O2 sustain up to 72h with minimal cell death. This is due to the fact that hypoxia-inducible factors activate anti-apoptotic responses.
In addition, neurons are naturally exposed to hypoxia (brains physiologic pO2 is fairly low, considering). I would recommend you use between 0.2 - 0.5 % O2 (mind that bellow 0.2 you're in anoxia). And determine a curse as suggested here before.
Finally, cycloheximide and hypoxia concomitant exposures have to be carefully determined as you may induce necrosis on your cells.
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