I have been trying to detect the extent of apoptosis in adherent cells treated with doxorubicin. I am using flow cytometry-based detection with Annexin V-FITC/PI and getting ~ 30-40% population positive for only PI (upper left quadrant) added to another ~ 40% in upper right. Morphologically the cells do not look like necrotic, so this is weird to get such huge population in upper left. My cell pellets look red and I am suspecting its basically dox contributing towards the red fluorescence, rather than necrotic/late apoptotic population. Does anybody have similar experience? I found people routinely using such kits for dox-treated cells. Can I get some alternative to replace PI that would not show emission in similar range as of Dox? Can 7-AAD be a better alternative? Any suggestion is appreciated. Thanks!
7-ADD is certainly a better choice given the Dox mix results! The 7-ADD has also been used more often!
Hi Paulami,
Of course 7-AAD is a good option for PI but if you still need to use PI then try to optimise its concentration as it often leads to such results when used in high concentration.
sometimes improper cell fixation prior to staining also creates such issues!
Best luck!
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