The whole thing doesn't make sense to me either. Perhaps it is the Hokey Pokey Now we got the thing with the parameter labels going quite well, but what is the sample, is it gated, did you use offset values for the log amps, what's the voltages....... Compensation is a wonderful thing - best left untouched. It allows a lot of things to be squeezed in boxes. In the absence of knowing what the sample is I am free to speculate about the biological role of CD3 negative but CD4 and CD8 positive cells. Where are the low CD3 positive cells in the comp 2 series? Is the poor CD3 separation down to lack of alignment or is it lack of antibody or..... Questions questions questions May the flow be with us - or was it the force ? Regards Gerhard PS I guess you mixed up the order of the 2nd set of over-compensated pictures. PPS I thing my switch is broken already -----Original Message----- From: Andrew Oberyszyn [SMTP:oberyszyn.2@osu.edu] Sent: Wednesday, January 02, 2002 5:07 PM To: Cytometry Mailing List Subject: Compensation question Hi Flow people! First let me wish everybody Happy Holidays and keep up the great work on this newsgroup! I ran some 3 color samples (CD3 FITC/CD4 PE/CD8 PE-Cy5) on my Beckman Coulter Elite today and I saw something that didn't make sense to me. I've attached three slides of the histograms that I got. I ran the single color positives and compensated so that the mean values where correct (see Slide 1, compensation settings #1). I then ran the samples (example of one on slide 2). There where 2 populations that didn't seem right to me (looked like they where undercompensated). I increased the compensation so that it "looked right". I then ran the single color controls again using the increased compensation values (slide 3). The mean values I got seemed to be overcompensated. I apologize for being somewhat ambiguous in the descriptions above (I'm trying to at least get out of here on time today!) but the histograms in the attachment are more informative. I've done three color work before but have never come across this problem. The user says that the antibodies are the same (i.e. not different lots within an antibody throughout the samples). I realize that alot of people are on vacation already and responses to this might be sparse but any help would be greatly appreciated (Mario? Help!) Thanks in advance! Andy (:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)-(:) Andy Oberyszyn, M.S. The Ohio State University Analytical Cytometry Laboratory 416 Comprehensive Cancer Center 410 West 12th Avenue Columbus, Ohio 43210 Tel: 614/292-FLOW(3569) Fax: 614/292-7335 E-Mail: cytometry@osu.edu (-):(-):(-):(-):(-):(-):(-):(-):(-):(-):(-):(-):(-):(-) "What if the Hokey Pokey is really what it's all about?!?" << File: Slide1.JPG >> << File: Slide2.JPG >> << File: Slide3.JPG >>
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