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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