Hello,
I am trying to measure caspase activity in CHO cell samples using the Flurochrome-labeled inhibitor of caspases assay (FLICA). I want to look at ethanol (or another checmical) induced apoptosis.
Does anybody have a protocol for this. Or could let me know for how long to leave the CHO sample in ethanol and what percentage.
I was originally trying to use heat- however we are not picking up any caspase activity in the sample.
Using Chemometic NC-3000
Thank you!
Caspases are central to the execution of apoptosis. Their proteolytic activity is responsible for the demise of cells in many physiological and pathological states. The initial activation of a caspase in a caspase cascade is a crucial event that determines whether a cell will ultimately undergo cell death. Although each cell contains a number of different caspases, only a small subset may be required for apoptosis in response to a specific stimulus.
Although ethanol is toxic to the cultured cells and commonly used for fixing cells, low concentrations of ethanol has recently been reported to induce apoptosis in cultured cells in vitro. You can cultred your cells of interest in medium containing 1mM ethanol for 12 hrs to induce upstream as well as down stream caspases.
In addition, I would suggest you to use H2O2 (25 uM or 50 uM) for 3 to 6 hrs to induce caspases activity in your cell of interest. Hope both exposure protocol may work to induce caspases in cultured CHO cell.
Good luck
Hello J. Cl
I would recommend H2O2. H2O2 is a broadly effective apoptosis inducer, but the dose range will differ with cell type. For cell lines, a low dose is required, and the exposure time must be reduced compared to primary cells to avoid cell death primarily by necroptosis or necrosis.
Please refer to the article attached for more information.
Article Is Hydrogen Peroxide a Suitable Apoptosis Inducer for All Cell Types?
Best.
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