We have been trying to flow sort live CD45+ cells from mouse intestines. One major issue is that these live cells continue dying after sorting. This is very critical for our following experiments that need at least 90% live cells for ~ an hour before we break the cells.
Thanks a lot for your suggestions.
Check your sheath fluid. I once had an ongoing problem that was the result of a new brand of sheath fluid being used. The problem can be the preservative(s) used in commercial products that can kill some cells. We had high viability sorts (we sorted live cells based on PI staining) and trypan blue staring afterwards looked good. We spun down cells once and there was just a little sheath left in the tube, but if the cells were cultured without much further dilution it was enough to kill all the cells the next day. If you suspect the same, try a sort with just PBS, and you can check the toxicity of the sheath by added a little to cultures etc.
Other possibilities are to try and sort into medium, check the room temperature, reduce the flow rate, make sure the sort streams direct cells into fluid and not the plastic walls of the collection tubes.
Thanks for your suggestions. I checked with my lab and they used just PBS as the sheath fluid. So there is not supposed to be any preservatives in it. But that does raise a question: will adding some FBS (say 2%) in the the PBS as sheath fluid help the cell viability ? The sorting temperature is 5 degree C and the flow rate is 4000 cells/sec. We will make sure the sorted cells go directly to the fluid of collection tubes next time.
Adding protein to the sheath is not a good idea. Will cause problems. I use 0.3% BSA in both the sample tube and collection tubes. 0.5% FBS works as well.
if these are large cells (macrophages) then maybe use a lower pressure/larger nozzle and slow down.
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