Hi!
I would like to discriminate dead cells from my population using LIVEDEAD fixable dye. In principle it stains amine residues in both live and dead cells, but because of cell membrane permeabilization dead cells have at least 50-fold higher MFI. I perfomed staining in this setup:
a) Unstained cells
b) stained untouched cells - should be alive
c) temperature killed cells - sholud be dead
d) mixed sample 1:1 alive and dead cells
e) FMO for LIVEDEAD (in case of presence of other staining)
Sample B histogram is moved towards figher FI than UC/ FMO - which is consistent with manufacturer description - what makes gates based on UC/ FMO useless. Positive (dead) population -sample C- is clearly of a very high FI. In order to separate negative (live) from postive population I set the gate on mixed sample where I can see clear two populations and marked the one with lower FI as LIVEDEAD negative = live cells. Does it sounds reasonable for you? Do you have other methods for proper gate setting with LIVEDEAD stains?
I would probably use a positive control (boiled cells) or even better a mixed control just for setting up properly the laser voltages in order to see a nice separation between my negative and positive population. However, boiled cells are "extremely" positive, at least for propidium iodide staining. The dead cells of your actual experimental sample will probably have a lower FI, compared to the boiled cells. To sum up, I would suggest selecting one of your actual experimental samples that contains both alive and dead cells to set the right gate. During your analysis, It is also important to check if that gate makes sense for all of your other samples, because you might need to readjust it slightly.
it would be nice if we could see the your figure. without looking the distribution of the cells, interpretation is difficult from the explanation.
As you requested I'am posting histograms. As far as I am concerned gating on mixed population (live and dead cells) is good.
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