How "huge" are the two populations (CD4+CD3- and CD8+CD3-)? Do they have well-defined boundaries in the two-parameter dot plot? Are they also highly fluorescent for the channel you don't use, if any? Upon activation, T cells could internalize CD3 rapidly, leading to some cells with dim CD3 staining. In case of relatively high background for whatever reasons, those cells would be regarded as CD3 negative. Moreover, for some mysterious reasons, lengthy and harsh isolation procedures may also result in loss of CD3 staining. Dead or dying cells can definitely look funny. So, you might want to put your isolated cells in culture for 12 hours to check whether they really survive. If they do, stain CD3 again before seriously considering the issue of immature thymocytes. Hope this helps. Hai Qi -----Original Message----- From: Dr. Philippe Stock [mailto:philippe.stock@charite.de] Sent: Thursday, October 26, 2000 4:29 AM To: cyto-inbox Subject: FACS naive mouse Hello, I am pretty much a FACS beginner. I have a question, which nobody has been able to answer so far. Every time I do a FACS scan from peribronchial lymph nodes (naive BALB/c mice) and stain it for cd3, cd4 and cd8 cells I see a huge population of cd4pos/cd3neg cells, as well as cd8pos/cd3neg cells. Does anybody know what types of cells these are? I have to admit that isolating PBLN from naive mice is rather tricky, usually leaving me with extracting the entire hilus and meshing that. Could these cells derive from Thymus (which I am pretty sure to extract beforehand) or from oesophagus? Thank you very much, Ph. Stock
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