PI, CdCl2 and cytotoxicity - (Apr/08/2014 )
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,
There are a number of options - some of which you could test - does CdCl2 increase autofluorescence? Is it actually killing your cells? - try doing a trypan blue stain and visually counting. Maybe CdCl2 enhances clumping for some reason? Are you sure that your gates are correct? What are your controls?
Thanks for the comments,
I will actually perform a microscopy visualization to complement the flow cytometry data. The problem is that cytotoxicity generates big debris (or not? remains to be confirmed) in my gate so it increases the event concentrations: this is what i undoubtedly observe. I am sure that my gates is correct. My control is the vehicle control (same solvent concentration not supplemented with CdCl2)...
Did the smaller debris increase as well ? Those woth a small FSC/SSC and thus concentrate in the lower left corner of the plot. Depends on the cell size and the settings (voltage) though on how it is displayed and if it is well separated from your actual cells.