The problem you are having is a consequence of the PHA/Ionomycin (sure is not PMA?) stimulation. Interesting enough, it does not happen in either CD8 or CD3 staining. Two alternatives are to gate on CD3+CD8- cells after stimulation or stain the cells with CD4 BEFORE stimulation with PHA/Ionomycin. We used the first approach in two studies bellow: Kallas EG, Gibbons DC, Soucier H, Fitzgerald T, Treanor JJ, Evans TG. Detection of intracellular antigen-specific cytokines in human T cell populations. J Infect Dis. 1999;179:1124-1131. Kallas EG, Reynolds K, Andrews J, Fitzgerald T, Kasper M, Menegus M, Evans TG. Cytomegalovirus-specific IFNgamma and IL-4 are produced by antigen expanded human blood lymphocytes from seropositive volunteers. Immunol Lett. 1998;64:63-69. Hope it helps Esper Kallas Em Quinta, 17 de Outubro de 2002, David Ritchie <dritchie@malaghan. org.nz> escreveu: >Any suggestions welcomed on below problem, > >We are tracking the cytokine profiles (by intracellular staining) produced >by CD4+ T cells in patients who have undergone allogeneic bone marrow >transplant (BMT). We are looking at cytokine production pre- and >post-stimulation of T cells with PHA/ionomycin for 4 hours. We have no >difficulty clearly identifying CD4+ T cells on the unstimulated sample, but >on the stimulated sample CD4 seems to disappear. CD3 is still clearly >evident. Has anyone seen this before? Is the cell fixing process used for ic >staining likely to interfere with CD4 expression or anti-CD4 binding? We >don't seem to be losing cells in the short culture period. > >Thanks for any comments > >David Ritchie >
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