Hi,
I'm trying to determine a sub-lethal dose of hydrogen peroxide to use for my cells (skin fibroblasts and keratinocytes) for subsequent respirometry experiments. I'm planning to expose the cells to varying concentrations of H2O2 for 1 hour and then using alamar blue to measure cell viability. In a protocol I've seen in a paper they remove the media containing the H2O2 and put fresh media with alamar blue on the cells for up to 4 hours for the reaction to occur and then read on a plate reader. My question is, do the cells recover from the H2O2 exposure during that 4 hour alamar blue incubation period? Should I find a different method to measure cell viability that does not involve a time delay for measurements?
The time of incubation after H2O2 exposure depends on your cell type. The cells will not likely "recover from the H2O2 exposure" because H2O2 induces a cell stress and will eventually kill the cells. The cytotoxicity may take between 4h and 24 hours depending on the cells and the dose of exposure. I hope this helps...
Good luck
Hi Michele,
Thank you for the answer, it is very helpful. I just want to make sure I understand one thing correctly. "The cytotoxicity may take between 4h and 24 hours depending on the cells and the dose of exposure." So depending on the cell type, cell stress --> cell death may take between 4 hours and 24 hours?
Yes that's right. Sometimes it may take even longer, but my experience of cell stress and apoptosis is mainly with tumor or leukemic cell lines and chemotherapeutic drugs, although I have also used H2O2 in some occasions. You have to first test a kinetics and dose-effect curve of cell exposure, then determine which is a good incubation time for you to get an average tox allowing you to see differential effects (an increase or decrease compared to control untreated cells) on your cells according to the dose of the stress agent. Once the conditions are fixed you keep the same assay all the time so you will be able to compare different samples and different experiments.
Good luck
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