Thank you to all that responded re: low # cells to analyze. (30 responses day one! I sure can pick the topic,eh?) I see from the varied responses I received I was waaaay too vague on what I was asking. So please allow me to re-post this query a bit more detailed. My friends are looking at chemokine expression in monocytes in CSF of MS patients. Typically their TOTAL WBC yield is 4-5000. That's it. They set up three 3 color tubes to look at CD3, CD14, and 3 chemokines.(cytometer= Facscan). The end result- they collect MAYBE 1000 total events/tube, get (on average) 100 monocyte events. Chemokine receptor expression is usually a SHIFT, not a clearly separated population. I guess I could feel better about analyzing 100 monocytes for chemokine expression if positives were very +/+ (quadrant2). But since you are looking at- what, 25-50 events, shifting across a marker; this just really makes me wonder. If you pick up ANY noise in your line- do you not worry? Is it because the FSC/SSC is so far over (from usual debris) for mono's that the gate can't possibly have erroneous events? My worry was even 10 "odd events" could inflate quadrant3 (-/-), thus affecting the percentage profiles in Quad's 1/2/4. Am I a worry-wort, or is this a legitimate concern? I also thought a slide (confocal or something along those lines) would make more sense than flow. (Thank you to everyone that sugessted confocal or CompuCyte LSC). I have since heard from several people who run bacteria and collect only 100-500 events and say their data is very tight. Is this because they have a very bright, very specific signal? To those that suggested additional markers: they cannot add any more (control)markers, because that would decrease the data collection even more. (damned if you do/damned if you don't!) Thanks again everyone- Bunny ******************************************************* Bunny Cotleur +*+ Bunny Cotleur Cleveland Clinic Foundation *+* 2001 Lester RD Neurosciences NC30 +*+ Valley City, OH 44280 9500 Euclid Avenue *+* 330-483-4800 Cleveland, OH 44195 +*+ bunny@cotleur.com 216-444-1164 *+* cotleua@ccf.org +*+ ******************************************************* When you do something, you should burn yourself completely, like a good bonfire, leaving no trace of yourself. (Shunryu Suzuki)
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