An optimized multi-parameter flow cytometry protocol for human T regulatory cell analysis on fresh and viably frozen cells, correlation with epigenetic analysis, and comparison of cord and adult blood

J Immunol Methods. 2013 Jan 31;387(1-2):81-8. doi: 10.1016/j.jim.2012.09.014. Epub 2012 Oct 9.


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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Cell Survival / genetics
  • Cell Survival / immunology
  • Cells, Cultured
  • Cryopreservation / methods*
  • DNA Methylation
  • Epigenesis, Genetic*
  • Fetal Blood / cytology
  • Flow Cytometry / methods*
  • Forkhead Transcription Factors / genetics
  • Forkhead Transcription Factors / immunology
  • Forkhead Transcription Factors / metabolism
  • Humans
  • Infant, Newborn
  • Leukocytes, Mononuclear / cytology
  • Leukocytes, Mononuclear / immunology
  • Leukocytes, Mononuclear / metabolism
  • Linear Models
  • Middle Aged
  • Polymerase Chain Reaction
  • Sequence Analysis, DNA
  • T-Lymphocytes, Regulatory / cytology
  • T-Lymphocytes, Regulatory / immunology*
  • T-Lymphocytes, Regulatory / metabolism*
  • Young Adult


  • FOXP3 protein, human
  • Forkhead Transcription Factors