Bunny If I assume that you are using a FACScan or FACSCalibur you are seeing it correctly. B-D in their infinite wisdom did not provide compensation between FL1 and FL3 nor did they provide for "negative" compensation. They rely on the fact that the compensation ratios are the same for FL1/Fl2 and FL2/FL3. They are close but not always (never?) perfect. It could be also that you have a fluorochrome that it not spectrally the same as fluoroscein or PE (R-PE I assume?). We don't always see it this much overcompensated in fact most of the time it does work well. DO other antibodies show the same problem. Suppose you could have a dichroic/filter problem. If not, not much to do except play with the PMT voltages and try to reach a happy compromise. Also just a note. You don't want to change the "brightness" of an antibody reagent by titrating it down. You need to use it at saturation. If you think it is too bright you must add some IDENTICAL unlabelled antibody to change the specific activity but not the antibody concentration. Larry Arnold At 10:59 PM 12/27/99 +0000, you 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) > Larry W. Arnold, Ph.D. Res. Assoc. Prof. Director, Flow Cytometry Facility Department of Microbiology and Immunology CB# 7290 University of North Carolina Chapel Hill, NC 27599 Phone: 919-966-1530 FAX: 919-962-8103
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