Hi everybody and thank to whomever will help me.
I am very new in flow cytometry so still understanding the basics. I am doing Annexin V - PI double stainging by flow cytometer in order to verify whether my patient-derived glioma stem cell line die after treatments and eventually how.
I selected the gateing strategy using the untreated control. Following the indications, I gated 10k events. To achive this resultes in the control, I needed to read a total of about 14k events.
When I analysed the treated sample, I saw a large increase of the events at the bottom left corner. Additionally, to gate 10k, I needed to read 37k total events. Anyway, the percentages of alive cells are quite the same in the two groups.
I don't think this kind of analysis is reliable, even counting the fact that through cell counting with Trypan Blue, I saw a strong decrease in the total amount of cells and a gain in the "blue" ones in the treated sample.
What am I doing wrong? How can I correct my analysis?
To make sure your gating and analysis is correct, I would include positive controls. Either an apoptosis inducer (for AV) or kill all cells (for PI).
I will add that you should also check your staining protocol. Adding a positive control will ensure your staining protocol is correct.
Even without the apoptosis staining, it is obvious in FSC/SSC plot that all of your cells are alive. So you didn't actually do anything wrong with your staining and gaiting protocols. I think that your problem is in treating protocols (probably the concentration of the drug was to low for the high count of treated cells ). But in future, in order to separate late apoptotic and necrotic cells properly, it is good to use the FMO positive controls (fluorescence minus one). Due to apoptotic bodies, Ann+PI+ double positive (late apoptotic) cells are hard to separate from Ann-PI+ (necrotic) cells. FMO positive controls mean that you stain one positive control only with Ann and the other only with PI. That will help you with the proper separation of late apoptotic and necrotic cells.
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