Quantitative analysis of Foxp3, T-bet, and GATA3 mRNA expression in sorted populations of CD4⁺ T cells. (A) Gating strategy for flow sorting mononuclear cells from the thymus and cord and adult blood. Profiles are gated for lymphocytes expressing CD4. Numbered boxes indicate the sorting gates. (B) RT qPCR for Foxp3 was performed using RNA prepared from sorted cell populations. Normalized Foxp3 mRNA levels were calculated relative to β-actin signals for each sorted population. (C) Expression of mRNA for T-bet and GATA3 in adult peripheral-blood CD4 T-cell populations sorted according to the gating strategy illustrated in panel A. Relative expression was determined by normalization to β-actin. Results are representative of 3 independent experiments.