Andrew, When we used to do compensation on some 4C work with PMBC harvests using FITC; PE; TR; and APC it seemed that even with setting comp. on all the appropriate single colors, once the actual sample was run the display was unsatisfactory. Our solution, while not perfect but allowing us to proceed, was to gate on the lymphocyte pop. only while running single color comp controls. The reasoning and actual empirical evidence being that the monos, Macs and other cell types were at varying levels, non-specifically staining for inappropriate probes. We would then open up the scatter gate and run our samples. This strategy yielded staining of subpops. that made sense to the biologists. It always helps to know what kind of cells you're running. If it is a cell line or some other homogenous pop. then all of the above is probably of little worth to you. Good Luck, Don Walker -----Original Message----- From: Andrew Oberyszyn [ mailto:oberyszyn.2@osu.edu <mailto:oberyszyn.2@osu.edu> ] Sent: Wednesday, January 02, 2002 9:07 AM To: cyto-inbox 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?!?" The information contained in this email is intended for the personal and confidential use of the addressee only. It may also be privileged information. If you are not the intended recipient then you are hereby notified that you have received this document in error and that any review, distribution or copying of this document is strictly prohibited. If you have received this communication in error, please notify Celltech Group immediately on: +44 (0)1753 534655, or email 'is@celltech.co.uk' Celltech Group plc 216 Bath Road, Slough, SL1 4EN, Berkshire, UK Registered Office as above. Registered in England No. 2159282
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