its likely to be due to the bright cychrome signal needs extensive compensation, in effect damping down the APC signal. Switch the reagents so that something less bright is coupled to cychrome and something brighter is on APC. And he should have more than a log shift for CD4-bio/SA-APC - are the reagents saturating ? and if its hardware compensation, check that its not over-compensated. good luck, Rachel ======================================================= Rachel M. Gerstein, Ph.D. Department of Molecular Genetics and Microbiology Graduate Program in Immunology/Virology University of Massachusetts Medical School 55 Lake Avenue North Worcester, MA 01655-0002 (508) 856-1044 (508) 856-5920 (FAX) > ---------- > From: Ayala Sharp > Sent: Tuesday, June 25, 2002 8:48 AM > To: Cytometry Mailing List > Subject: Quenching of CD4 - APC signal > > > Hi All > One of our users performs a 4-color staining on the FACSCalibur with the > Abs > detailed below. When he looks at the CD4-biotin-sav-APC positive staining > alone (in mouse thymocytes) there is a one decade shift on the log scale > of > the FL4 signal. When, however, he adds the other Abs, the staining of the > FL4 is nearly completely abolished and he can't focus on the CD4+/CD8+ > population. (Even when he performs his staining as follows: first > incubating > the cells with anti CD4-biotin, washing, adding sav-APC, washing, than > adding the other three Abs). > Any explanations ? Suggestions ? > Thanks > Ayala > > The Abs I am using are: > > CD8a Cy-Chrome conjugated Rat anti-mouse monoclonal Ab (Pharmingen) > > CD4 Biotin conjugated Rat anti mouse monoclonal Ab (Pharmingen) > > Streptavidin-Allophycocyanin (sav-APC) conjugate (Pharmingen) > > Rat anti-mouse CD24/HSA R- PE from Southern biotechnology Associates, > Inc. > > Anti-mouse TCR beta clone H57-597(FITC) from Southern biotechnology > > Associates, Inc. > > The problem is as you have seen inconsistent FL4 (CD4-APC) staining when > > combined with FL3 (CD8 Cy-Chrome) staining. > ------------------------------------------------------------------- > Dr. Ayala Sharp > Biological Services, > Weizmann Institute of Science, > Rehovot, ISRAEL. > FAX: 972-8-9344194 > Tel: 972-8-9342721 / 972-8-9342235 > E-mail: ayala.sharp@weizmann.ac.il > > > > >
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