Keith, are you not able to gate these cells out based on their scatter - lower FSC and higher SSC - or are they contaminating your population of interest? Candace Enockson --On Mon, Apr 17, 2000 10:18 PM -0400 Keith Bahjat <kbahjat@ufl.edu> wrote: > > I can say that in our culture systems following activation, we often find > cells of questionable viability which will bind any antibody you throw in > the tube. You need to make sure you have some marker that these cells are > negative for, like CD13, or F4/80. Something you would never expect to > find on a T cell. When you're in the habit of staining with 4 antibodies, > and looking for cells positive for all 4 antibodies, you're just asking > for trouble. > kb > > Keith Bahjat > kbahjat@ufl.edu > > > > on 4/14/00 10:03 PM, Qi, Hai at haqi@utmb.edu wrote: > >> >> It is not surprising. I believe you will see double positive cells, and >> they are primarily ACTIVATED T cells (this is potentially why you find >> they are bigger, assuming not too many doublets are there). Further, you >> may see T cells expressing different levels of B220. In my experience, >> this phenomenon is also not specific to cells in culture. Overall, I >> don't think B220 is really an absolute marker for B cells. Hopefully >> experts out there will comment on this. >> >> To gate out cell aggregates, I think you can plot pulse height vs width >> of your CD4/CD8 channel (several washes with ice-cold staining buffer >> should also help). >> >> Hai Qi >> Pathology, UTMB >> >> -----Original Message----- >> From: Olindo Assis [mailto:oamfilho@cpqrr.fiocruz.br] >> Sent: Wednesday, April 12, 2000 1:01 PM >> To: Cytometry Mailing List >> Subject: unexpected T and B phenotypes after culture >> >> >> We have been phenotyping splenocytes from different strains >> of mouse before >> and after stimulation with bacteria (H. pylori) antigens. We >> are basically >> labeling the cells using anti-CD4, anti-CD8 and >> anti-CD45(B220) PE from >> SIGMA. Labeling procedures were done in separated tubes. >> Frequently we have noticed that the sum of CD4 + CD45 + CD8 >> cells from the >> unstimulated cultures give us a number over 100%. >> It is interesting to observe that this phenomena is observed >> only in >> unstumulated cells. Cultures in the presence of H. pylori >> antigens does not >> show this problem. Moreover it was observed for all mouse >> strains evaluated >> independent of the age. We have used Balb/C, C3H/HeN and >> C57/BL6. >> It is interesting to observe that these overestimation is >> observed only in >> the region corresponding to larger cells. We first suggested >> that this could >> be due to the presence of doublets of CD4 and CD45 cells. >> However it could >> be also due to the presence of cells co-expressing both >> phenotypes. >> In order to solve this we are currently evaluating the >> presence of double >> labeled cells using anti-CD4 FITC and anti-CD45 PE in the >> same tube. >> However, if we find double labeled cells we still have the >> question whether >> they are representing doublets or bi-phenotypic cells. >> Does any one have experience of phenotypic analysis of T and >> B splenocytes >> after control cultures? Any suggestions? >> Is that possible that CD45 (B220) could be a marker for >> cultured CD4 cells? >> >> >> Any help is appreciated, >> >> Olindo >> >
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