--On Wednesday, April 17, 2002 2:29 PM -0500 Renee Read <rdread@artsci.wustl.edu> wrote: > > I am a graduate student and I've been using flow cytometry to study > the cell cycle in Drosophila cells. I dissociate cells from tissue > samples and stain for DNA content with Hoechst 33342 and it works > great. However, I've been trying to also use GFP as a marker in these > assays and I've found that the Hoechst flouresces in the GFP channel and > obscures the GFP signal to the point where compensation can't fix the > problem. We are using a Cytomation Moflo mahcine and we are using the > UV laser for the Hoechst and the FL2 detector for the GFP. We've tried > shutting off the UV laser and the Hoechst background is still picked up > in the GFP chanel, indicating the problem is with the FL2 detector. The > flow technician isn't sure how to fix the problem. Do you guys have any > suggestions on how to look at cell cycle and GFP flourescence in live > cells? Thanks > > Renee Read Hi Renee Are you saying that you are just using a single laser (UV) and exciting both GFP and Hoescht from it? I too have been looking at Hoescht 33342 and GFP in Drosophila cells. I use a MoFlo, configured to excite first with 488nm in the first pin hole (with a 530/40 filter on the detector to pick up GFP), then UV in the third pin hole (with a 450/65 filter on the detector to pick up Hoescht) What we have found is the cell debris autofluorescence will usually be picked up by the 530nm detector, but is easily gated out. Let em know if I can help further. I have some dot plots of the sort of thing we are seeing. Richard -- Richard Grenfell Laboratory of Molecular Biology Medical Research Council Cambridge rlg@mrc-lmb.cam.ac.uk
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