Flow Cytometry Questions - debris removal and G2/M visualization (Dec/08/2005 )
I am starting to do some flow cytometry experiments to look for cell cycle arrest with a cytotoxic natural product. Mostly using LnCAP cells, but also some T-47D experiments.
First, can anyone suggest a good method for removing cellular debris? To stop my LnCAP cells from clumping I need to either trypsinize quite a while, or else pipette vigorously. Either way, I get a bit of debris I would like to remove.
Second, I'm not seeing much of a "peak" for G2/M phase, despite using cells at various degrees of confluence. Is there anything I can do to bring this out? Would a different dye- or more concentrated dye - help? Or longer incubation? I'm currently using Vybrant Orange from Molecular Probes.
Thanks in advance.
Hmm, maybe I posted too soon. The problem seems to have taken care of itself.
The problem was not so much with the cytometry as with the hematacytometer counting. If I count first, then plate out for a day, I can suck off the debris, then very briefly trypsinize. Now the cell cycle plots look beautiful.