Hi Renee, You can be sure that you aren't getting any 488nm excitation of the Hoechst by running cells with and without the dye. If there is no shift then but there is when you open the UV laser what you are probably seeing is emitted fluorescence from the UV-excited Hoechst being detected through the primary laser pinhole. We have had similar problems. Check that your second laser is optimally focussed first of all and use a relatively low laser power (you dont need a huge amount to get a good Hoechst signal, we operate at no more than 60mW but that is mainly because of paranoid users, I think you can get away with less). Another thing you can do is to custom make a pinhole assembly so that the distances between lasers are greater (remember to change the time delay) Derek On Fri, 19 Apr 2002, Renee Read wrote: > I recently posted a call for suggestions on how to use a > Hoechst/GFP double stain in a cell cycle analysis and I want to correct > an error in my post and address some problems. First, I erred when I > said that we tried shutting off the UV laser and the Hoechst background > is still picked up > in the GFP chanel. Being a fly geneticist and not a flow expert, I > misunderstood the problem. The spillover of the Hoechst into the GFP > chanel goes away when we turn off the UV source. We are trying to > distinguish GFP positive from GFP negative cells at the same time that > we are looking at Hoechst staining, and we have trouble with the > background from the Hoechst obscuring that distinction. The cells do > express GFP from a genomically integrated reporter, but not at a really > high level. So perhaps the problem really is with the cells. > The instrument settings we've been using are as follows: The MoFlo > has the 20 X 60 micron 488nm beam in pinhole #1, and the 20 X 60 micron > UV beam in pinhole #3. Emission path filters are HQ510/20 in FL1 for > GFP and 440/40 in FL8 for Hoescht. 488nm output has consistently been > 100mw and we have varied the UV output from 20 to 100mw to obtain less > encroachment. > > ************************************************************************ Derek Davies Voice: (44) 020 7269 3394 FACS Laboratory, FAX: (44) 020 7269 3100 Cancer Research UK, e_mail:derek.davies@cancer.org.uk London Research Institute, mobile: 07790 604112 44 Lincolns Inn Fields, London, UK. Web Page: http://sci.cancerresearchuk.org/axp/facs/davies/index.html In tenebris lux *************************************************************************
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