Multi-parameter flow cytometry analysis of T regulatory (Treg) cells is a widely used approach in basic and translational research studies. This approach has been complicated by a lack of specific markers for Treg cells and lack of uniformity in the quantification of Treg cells. Given the central role of Treg cells in the inception and perpetuation of diverse immune responses as well as its target as a therapeutic, it is imperative to have established methodologies for Treg cell analysis that are robust and usable for studies with multiple subjects as well as multicenter studies. In this study, we describe an optimized multi-parameter flow cytometry protocol for the quantification of human Treg cells from freshly obtained and viably frozen samples and correlations with epigenetic Treg cell analysis (TSDR demethylation). We apply these two methodologies to characterize Treg cell differences between cord blood and adult peripheral blood. In summary, the optimized protocol appears to be robust for Treg cell quantification from freshly isolated or viably frozen cells and the multi-parameter flow cytometry findings are strongly positively correlated with TSDR demethylation thus providing several options for the characterization of Treg cell frequency and function in large translational or clinical studies.
Bibliographical noteFunding Information:
We thank Dr. Elizabeth Goetz for obtaining cord blood for this study. We thank the University of Wisconsin Carbone Cancer Center Flow Cytometry Laboratory staff, in particular Ms. Kathleen Schell for her patience, generosity, and expertise. This study was supported by Midwest Athletes Against Childhood Cancer Fund and the University of Wisconsin Institute for Clinical and Translational Research , funded through an NCRR/NIH Clinical and Translational Science Award , 1UL1RR025011 (both awarded to CMS).
- Cord blood
- Flow cytometry
- T regulatory cell