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?

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