CFSE staining and unlabeled cells

Hi flow'ers community,

I performed CFSE staining of peripheral blood lymphocytes. I followed the 
protocols found in the literature, that is to say suspension of cells in PBS 
at a concentration of 10^7 cells/mL, staning with 10uM CFSE, incubation for 
10 minutes at 37°C, washing with complete medium and plating. I stimulated 
cells with cytokines such as IL-7 and IL-15. At day 6, quite surpring, I 
found a consistent proportion of cells on the first decade of fluorescence 
adding to proliferating cells that diluted CFSE; I'm not sure they were 
proliferating cells at high rate, since they were found also in the 
unstimulated control. Have you got any suggestions? Have I to increase the 
concentration of CFSE or diminish the cell density at the time of staining?

Thanks to all,

Enrico

Flow Cytometry Unit
Dept. of Biomedical Sciences
University of Modena and Reggio Emilia
41100, Modena, Italy

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