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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