have had same problem with drosophila. Solution is simple. Put a density filter in front of the GFP detector. This will correct the "Hoechst contaminating fluorescence" leaving your GFP signal intact. The problem is not the detector but the sample itself (but not all samples are like that- it might be the protocol). Also when aligning the instrument make sure you use 488nm beads and UV beads which only fluorescence in their specific channels - If you have the same configuration as I have the slot for the density filter is before all the pmts off the 1st laser -so you will have to increase the SSC signal. Once you get this working you'll find it's a very nice assay. hope this helps- if you come up with another solution let me know. Regards Ann At 14:29 17.04.02 -0500, you 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 >Department of Molecular Biology and Pharmacology >Washington University School of Medicine >660 S. Euclid Avenue >St. Louis, MO 63110 >lab: (314)-362-7797 >fax: (314)-362-7058 >rdread@artsci.wustl.edu
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