My PhD student is working on phytochemical-induced cell death in cancer cell lines and we need to establish if the cell death is apoptosis or necrosis etc. As our budget is dwindling, does anyone know of cost effective ways of measuring apoptosis? We have tried YO-PRO-1 staining but it just won;t optimise for flow and the signal from microscopy is poor. Mnay of the kits available for Annexin etc are likely out of our price range for the number of repeates we need to do.
Advice greatly accepted!
JB
Since cost is your major issue, I would go low-tech and use a basic cell death assay where your readout would be absorbance, which you could measure using a standard plate reader. The reagents are cheap and one kit would last you a long time. Now coming to your need to distinguish between the different kinds of cell death, you could do rescue experiments as described below.
1. Cells + phytochemical: Cell death
2. Cells + phytochemical + zVAD: Complete rescue if the manner of cell death is apoptosis. Incomplete rescue if it is a mix of different kinds of cell death. One caveat is that caspase inhibition could switch apoptotic cell death to necroptosis.
3. Cells + phytochemical + Nec-1: Complete rescue if the manner of cell death is necroptosis. Incomplete rescue if it is a mix of different kinds of cell death.
4. Cells + phytochemical + zVAD + Nec-1: If you see complete rescue here but incomplete rescue in 2. or 3., what you are observing is a mix or a switch. If you see incomplete rescue here, you have some necrosis.
I am linking a colleague’s paper where they have used this strategy to study cancer cell responses. They used CCK-8 assay but MTT should work.
https://stm.sciencemag.org/content/8/339/339ra70/tab-pdf
Propidium iodide is dirt cheap and highly standard for late apoptosis/secondary necrosis. Combine it with a zVAD condition as Julie mentioned and you have a pretty great apoptosis assay. Should work on flow and microscopy, and in some cases even a plate reader but I've had less luck with that.
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