I would put my money on small aggregates, doublets or triplets. Take a look down a microscope. Removing calcium or adding some EDTA may help prevent this. How does the CFSE brightness compare to small lymphocytes? I've seem this sort of thing before which is exacerbated by staining with CD3 antibodies for some reason. Using Ab serum probably makes it worse. Simon Monard FACS Lab Manager Trudeau Institute Saranac Lake NY12983 Ph 518 891 3080 X352 >>> "Da Silva, Marcos" <Marcos.DaSilva@umassmed.edu> - 12/11/2000 4:40 PM >>> Colleagues, I've been working for the last months in developing a system to evaluate proliferation of human T lymphocytes in response to different antigens and mitogens. I am using CFSE as the proliferation marker, and CD3 (APC), CD4 (PE) and CD8 (Cy-chrome) as the fluorescent conjugates. I've been concerned with the fact that, after 5-7 days in culture, the blast population usually presents with 10 to 20 percent of CD3 cells that stain positively both for CD4 and CD8. This does not happen when I gate and analyze the small lymphos. It does not seem to make sense, comparing to the "in vivo" lymphocyte proliferation. Would it be an artifact of cultured rapidly proliferating cells? Could it be due to Fc-receptor binding of the fluorochromes, with my blocking (with AB serum) being inneficient? Thank you, Marcos da Silva Pediatric Immunology lab University of Massachusetts Medical School - Worcester - MA - USA e-mail: Marcos.DaSilva@umassmed.edu
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