I am working on A549 cells with an anticancer compound. I am planning to study the mechanism of cell death in those cells (apoptosis or necrosis).
Since, I am using flow cytometry, I want to use cells which are positive for apoptosis and necrosis dye.
For apoptosis, I plan to use doxorubicin for 3 hr or sutent for 24 hours.
I wanted to use doxorubicin for necrosis as well with 24 hour treatment. But, I was not sure whether that would work. I would greatly appreciate if I can get some suggestions for necrosis induction. Also, if my strategy if correct.
I am using the following dyes:
1. Apopxin - apoptosis
2. 7-AAD - Necrosis
Regards,
Pratik
It is not necessary to use special positive controls because cells cultured always have some baseline amount of apoptosis and necrosis. But if it is important in your experiment staurosporine or.. yes ... doxorubicine are good inductors of apoptosis. And some oxidants can be used to induce necrosis. H2O2 may be not a good choice it can induce both apoptosis and necrosis, although necrosis is more often.
In this review t-butylhydroperoxide is suggested to induce necrosis for positive control for flow cytometry https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3874588/
Or TNF-alpha at high concentration (higher then physiological concentration) can be used to induce necross in carcinoma cells.
Thanks.
For 7-AAD positive cells, I was thinking of using cell-permeabilizing reagent like 0.1% triton X-100 and 0.1% sodium titrate. They worked well for PI staining study for DNA cell cycle analysis.
You think that would work for necrosis as well. I mean, I just need to have positively stained cells for the dyes, right?
7-AAD binds to DNA like PI, it allow one to differenciate viable/dead cells or it can be DNA marker. So if cell is viable and has undamaged membrane this reagents will not stain it. Yes, cell-permeabilizing reagents (patricularly triton X-100) will produce 7-AAD positive cells as well as PI positive cells.
Yes positive control is about obtaining positively stained cells for the dye.
Triton X-100 is quite often used to show positive necrosis control.
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