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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