Measurement of the incorporation of radionuclides such as ³H-thymidine is a classical immunological technique for assaying T cell proliferation. However, such an approach has drawbacks beyond the inconvenience of working with radioactive materials, such as the inability of bulk radionuclide incorporation measurements to accurately quantitate T cell divisions, and an inability to combine proliferation analyses with simultaneous evaluation of the expression of cellular markers in divided cells. By labeling T cells with reactive dyes such as CFSE, Celltrace Violet, and others that are partitioned equally between daughter cells at each cell division, one can relatively easily track generations of proliferated cells and their expression of various molecules by flow cytometry.

FoxP3⁺ regulatory T cells (Treg) are critical mediators of immune tolerance and evaluation of their functionality is an important step in characterizing many immune models (Rudensky, 2011). Classically CD4⁺ Treg and conventional or “responder” T cells have been isolated based on their surface expression of CD25 (Treg: CD4⁺CD25⁺, Tresp: CD4⁺CD25^-). However, we and others have noted that populations of CD4⁺CD25^- cells express the FoxP3 transcription factor and have suppressive function. Therefore we have utilized the transgenic FoxP3-EGFP mouse to facilitate live purification of suppressor and responder populations based on EGFP (and thus FoxP3) expression. Here we present our adapted protocol for assaying regulatory T cell suppression of Celltrace Violet-labeled responder T cells.