live/dead discrimination - (Aug/19/2009 )
how do you discriminate between live and dead cells in a cell suspension by flow cytometry?
in your experience, is it better PI, PI+Rnase , 7-AAD or Trypan Blue ? and wich concentration of the dyes are the best?
7-AAD worked best for me. Although I forget the concentration, I'm sure you can find it somewhere.
Generally speaking, most of your dead cells will have high side scatter and low forward scatter. Most dead cells and debris can be gated out that way. If you're new to FACS, try a few experiments with 7-AAD and pay attention to where those cells end up on SSC vs FSC. Although in some cases you may have to use 7-AAD to be sure you're only looking at live cells, usually you can obtain perfectly good data just by gating based on forward and and side scatter, without staining with 7-AAD.
Trypan blue is an extraordinarily good quencher of fluorescence. You will definitely want to avoid this!
PI by RNAse is for doing cell cycle analyses. RNAase is added to clean up the results.
Both PI and 7-AAD are effective. 7-AAD is an easier dye to work with if you are doing multicolor analysis because it has less spill into the other channels