I was glad to see your comment on B220. We are using B220 in various experiments - on IEL, pyer's patches, lung associated lymph nodes, and spleen, control and infected and we see all sorts of B220+ populations, dim and bright. I am not very experienced with this marker and need all the suggestions this site can offer. Candace Enockson Medical University of South Carolina --On Fri, Apr 14, 2000 9:03 PM -0500 "Qi, Hai" <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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