I am incubing cells with CdCl2, then i stain my cells with PI in order to test the viability.
On my acquisition gate, i acquire 5000 events, that corresponds to autofluorescence of viable cells + PI-positive events (=dead nucleated cells).
However, in the cases of cytotoxicities, the gate counts 5000 events in less volumes (= more density of events). This is disturbing me because normally this gate corresponds to my population of cells, so only the viability should be changed, not the total concentration....
On one hand i may acquire two events coming from one original cells, on the other hand this is weird because i am excluding debris so cellular debris not nucleated should not appear on my gate, unless they are very big debris that can be counfonded with autofluorescence of viable cells. Moreover, when i look the morphology of the populations, in case of cytotoxicity, they are much more dispersed than the controls, they gained in both FSC/SSC and the cloud of dots is dispersed.
Any ideas for the increase of density observed?
Thanks a lot in advance,