Dear Urban,

In my opinion, these kits couldn't separate late stage apoptotic and necrotic cells. Many papers label late apoptotic cells as AV+PI+ and necrotic cells as AV-PI+. Apoptosis is defined by the flipping of the phosphatidyl serine which is detected by AV when the membrane of the dying cells is still intact (PI-). However, if the PI or annexin V can penetrate dead cells which have damaged membrane, it is hard to say positive AV staining is due to detection of flipped phosphatidyl serine.

Personally, I think the staining only allow us to detect live, apoptotic and dead cells. I had been asked to plot late apoptotic cells.

Dear Kai Ling, thank you for your opinion. I believe exactly the same.

Regards

Hello Urban, is critical to have proper controls of untreated cells where you can see perfectly necrotic cells from normal cultures. If you set the position of Ann+PI+ cells and compare with your treated cells, a population of cells appears below the position of necrotic ones, with positive staining for Annex but less positive for PI. Those cells can be considered as Late apopotic, because they have less amount of DNA because endonuclease is already active. Hope it helps, best regards, Alberto

Dear Urban,

This article might be of your interest though it is not focusing on PI+AV. ! It explains autophagy, apoptosis and necrosis and what markers/assays could be use to distinguish these distinct forms of cell death.