I need to sort murine NKT hybridomas via TCRb and CD1d double-staining, but I also want to discriminate live from dead cells. I have been using Blue live/dead fixable stain by Invitrogen in flow cytometry applications with good results, I wonder if I can use the same dye for cell sorting? I've been advised to use 7-AAD but I wonder if fixable dyes render the same good results.
Wanted to update my question/answer because I am at BD sorter training this week and I asked this question today in class. Answer I received is actually quite obvious now that I think about it.
BD Trainer: "Break your sort into shorter 15-30min intervals and add 7-AAD to each tube prior to acquistion/sorting". In this way, your avoid this issue of slowly increasing 7-AAD fluorescence of your "LIVE" cells. Moreover, your "stained cells" cells should have better viability because they aren't inside the pressurized chamber (ARIA) for extended periods of time.
Fixable dyes would be absolutely fine. Same principles still apply and you should be good for sorting (as long as you don't fix the dyes).
I have sorted several samples using 7-AAD as it is very easy to use, but fixable dyes should do the job as well. Keep in mind that a FSC vs. viability dye plot can further help to exclude dead cells.
Also, try to exclude cell doublets. This can be achieved by pulse geometry gating (e.g., FSC-H x FSC-A).
Calcein AM is a very bright live stain that doesn't seem to affect the cells downstream. It disappears after a day or so.
What is your end goal with the sorted cells? Are they being injected into an animal or for in vitro use? Think about toxicity of the dye, if it is something you need to be concerned with, as well as if you need the cells to viable (not fixed) or not; that can obviously make a huge difference in which dye to use.
This may be a naive question, but over the course of a longer sort (>1hr) does maintenance of 7-AAD in the cell suspension result in an increase of 7-AAD fluorescence by viable cells, and consequently lead to exclusion using the originally set gate? I have used LIVE/DEAD Fixable dyes in my past sorting experiments with success, but with the known caveat that cells could be dying in the sort tube and would not stain brightly with this viability dye because it is no longer present.
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