Hi Xinjian Is all your staining done with directly conjugated antibodies? Assuming it is. Two thoughts: Could one of your Ig antibodies be cross reacting with the CD4 FITC antibody? Maybe the CD4 FITC is a different isotype or made in a different species to the other CD4 abs. To test this you could stain with your B cell markers, incubate with a bit of serum from the same species as the CD4 FITC Rat? for 10 minutes or so then add your CD4 FITC and see if your results are the same. Another possibility is that your are seeing T/B doublets, triplets etc. or Macrophage/lymphocyte doublets triplets etc. How is the scatter of the cell population in question? If the double positive cells are twice or thrice the size of a nornal lymph or are the size of a macrophage then suspect the clumping theory. I can't think of a reason wht one CD4 antibody would promote clumping and not others. Your mice haven't been injected with Ig, CD4 ab or FITC have they? What are they immunized against Best Simon Simon Monard FACS Lab Manager Trudeau Institute Saranac Lake NY12983 Ph 518 891 3080 X352 >>> "Xinjian Chen M.D., Ph.D." <xchen2@emory.edu> - 4/11/02 9:00 PM >>> Greetings to everybody I have recently obtained some intriguing flow data, as described below, and am looking for help to understand its nature. When I perform multi-color staining on spleen or lymph node cells of immunized mice with anti-CD4-FITC plus other antibodies, I can unequivocally identify by flow cytometry a subset (fifteen to 30%) of CD4+ T cells that are positive for B cell markers, including B200, IgM, IgD, MHC Class II, etc. However, if I use same anti-CD4 antibody conjugated to other flurochrome, such as PE or PerCP, than FITC for multi-color staining, the B cell marker-positive subset of CD4 T cells becomes no longer identifiable. Your comments will be greatly appreciated. Best regards Xinjian
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