Hi, Are you using single-color reagents to set up your initial compensations? You didn't mention that in your question. Assuming you are, try lowering the PMT sensitivity on one color or another to see if your compensation improves. Also, since you haven't done 3-color in a while, are you sure your optical filters are properly set-up? (my 2c., I am sure you'll get other helpful hints.) Art Roberts On Mon, 27 Dec 1999, Bunny Cotleur wrote: > > Flow masters- > > I am having a bit of a dilemma. I haven't done any 3-color work in many > many years. And frankly, I don't recollect this issue cropping up > before. What I am trying to do is fairly simple. I am multi- staining > human lymphocytes for various chemokine receptors simultaneous with CD4 > and CD8. I have the following mixes (set up by someone in the labs, > who's work I am extending): > > CD4-PERCP/CD8-FITC/CHEMOKINE*-PE > CD4-PERCP/CD8-PE/CHEMOKINE*-FITC > > (I am staining for several chemokine receptors, the exact ones not > important here). > > Now here is my simple problem that is dragging me down into absolute > madness. When I PROPERLY compensate Fl1/Fl2 > (I say properly,as I have followed Mario's web instructions TO THE > LETTER. Except for all the crazy math that is making my head spin :-) > ).... I get frustrated. When FICT/PE are comp'd fine, then PE is > smack in quadrant 2 looking at PE vs Fl3. So, OK- I comp FL2/Fl3. > Picture perfect. Go back to Fl1 vs Fl3. AAARRRGGHHH! Green is now > way overcomp'd to the X axis. Let's just get scientific and say really > SQUOOSHED down. I have been going back and for with this for days. > Different sample sources. Titering down the Ab's. I call BD and they > say maybe my instrument is "too sensitive". HUH??? Is that possible? > I thought that was IDEAL. IN the meantime, I also note that %CD8 FITC > is NOT equal to %CD8PE in the same sample. (Both sources=BD, both > used as they recommend. Both also titred down to 2ul, no difference). > Now I'm starting to feel that I need to surrender my "License to Flow" > due to acute ignorance. > MAYDAY! > (***white flag is flapping in the breeze**) > -- > > > Bunny Cotleur > > Cleveland Clinic Foundation > Neurosciences / NC30 > > 216-444-1164 > > > > > > ****************************************************************** > When you do something, you should burn yourself completely, like a good > bonfire, leaving no trace of yourself. > (Shunryu Suzuki) > __________________________________ Arthur Roberts Dept. of Medicine Robert Wood Johnson Medical School email: robertar@umdnj.edu phone: 732-235-7790 Fax: 732-235-7792
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