Hello everyone,
I am new to flow cytometry. In my experiment, I want to use single PI staining to detect cell cycle change and percentage of apoptotic cells. I used 70% ethanol to fix cells after different treatment and then did PI staining to do flow cytometry. My question is if I want to measure apoptotic cell population, which part of pulse width versus area plot I should choose to get the data? The software I used is FlowJo.
There is a nice protocol here -
http://cancer.ucsd.edu/research-training/shared-resources/flow-cytometry/protocols/Pages/apoptosis-with-pi-only.asp
I will like to read this protocol too..But I am unable to open the link. Could you please resend the link? Thank you in advance
By doing the PI staining after 70% methanol you would be able to see the cell cycle profile in the FL2 channel (PE channel).
You can see the apoptotic versus live cells in the FscxSsc plot. Then you do a gate for live cells, inside of that duplets exclussion (FL2-W vs FL2-A). After all this, you should be able to see a nice cell cycle profile of your live cells in the FL2-H or FL2-A channels. If you include the all apoptotic cell population (live + dead) for analysis you may include major artifacts that may not allow you to do a proper cell cycle analysis. to have a more pretty cell cycle profile you may leave your samples fixed and stained for several days (protected from light and at 4ºC) and then acquire the samples.
The first couple of supplemental figures for this paper may help you:
http://emboj.embopress.org/content/embojnl/29/14/2407/DC1/embed/inline-supplementary-material-1.pdf?download=true
http://emboj.embopress.org/content/29/14/2407.long#sec-26
Here PI staining distinguishes the G1 population, G2 + mitotic cells and the sub-G1 component. PI staining is not so great on it's own to clearly distinguish S-phase, you could try BrdU incorporation for this and pHH3 for the mitotic cells.
You could think about using Z-VAD as a control in your experiments to show any sub-G1 is due to apoptosis.
Good luck with you experiments,
Helen
Actually you can not distinguish apoptotic cells using FscxSsc plot. You can just eliminate debri from your samples. You can not distinguish these apoptotic cells just using PI as well.
Dear all,
Thanks for you suggestions. I will try to check apoptosis using PI/Annexin V staining.
Thanks and I am still open for further suggestions.
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