This might be a long shot but I am going to try it!
I am working with primary neural cells. I have a live cell stain florescent that marks for caspase 3/7 cleavage. I am looking to do a proof of concept/control by inducing apoptosis in the cells to show that the dye is working as expected. In my literature review I found two primary methods to induce apoptosis One involving oxygen glucose deprivation (OGD) and the other H2O2 ROS stress.
My lab does not have the necessary set up or materials to do OGD so that is more or less out. H2O2 seems like a logical answer however their are a couple of problems.
Ideally the dye should be present during the injury/stress period to best capture the caspase 3/7 cleavage, however I am concerned that H2O2 will interfere with the dye (since many fluorescent dyes are sensitive to oxidation). Most studies use a 24 hour H2O2 injury period after which cell viability is measured and shown to be seriously decrease. My thought is to reduce the injury time - removing the H2O2 and adding the stain before 24 hours - but I can't find much research about the time dependent response to H2O2 injury. Does anyone have experience in this area or an idea of how long I should leave the H2O2 in? The trick is to add the dye before the cells get thought the apoptosis cascade but leave them in the H2O2 long enough to induce apoptosis events.
Alternatively, I have considered temperature stressing the cells by placing them in a warmer or cooler environment however I have not been successful in finding research or protocols to support this method. I have to be careful that the cells don't just become necrotic (because caspase 3/7 wouldn't be cleaved).
My only other thought is to put the cells in PBS instead of media for a period of time. I am not a molecular or cell biologist by training (engineer here) so maybe this is somewhat of a silly idea but would this potentially induce apoptosis? My thought is that the cells would be nutrient starved but I don't know if that would have the effect I am looking for.
If anyone has thoughts on any of the questions or ideas or has a suggestion for a better approach let me know! I have doing some pretty extensive literature review but I keep coming up with the same things, ODG and H2O2. Unfortunately as I said, we don't have glucose free media and definitely don't have the ability to deprive the cells of oxygen. Since this is not the primary focus of the research I am hesitant to spend money on specialized media or other equipment if it can be avoided.
Thank you for your time and any advice you have to offer!
H2O2 induced cell death is not very reproducible, perhaps could you use another way to induce programmed death in your neuronal cells by adding let say 100 µM Buthionine sulfoximine which blocks glutathione synthesis and leads, at least in our hands, to cell apoptosis within 24-30h of treatment.
Dear Cat J Hershey ,
Doxorubicin is a well-known inducer of apoptosis and should work as a positive control in concentrations between 0.5–5.0 μmol/L in your assay. (https://www.nature.com/articles/1206366, https://www.sciencedirect.com/science/article/pii/000629529390512U?via%3Dihub and https://www.mdpi.com/2072-6694/11/5/702)
Good luck - CL
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