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)
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