Quantitative analysis of gene expression in Th2-skewed cells sorted by CRTH2 or IL-4 expression. (A) Three-day cultures of CRTH2⁻ and CRTH2⁺ cells were stimulated (5 hours) or not with PMA and ionomycin and RNA extracted for quantitative real-time RT-PCR (see “Materials and methods”). Transcript levels are expressed as percent of the housekeeping control, PPIA. Means ± SEM of 5 experiments. *P < .05; **P < .01; ***P < .001 relative to CRTH2⁻. (B) IL-4⁺ cells were enriched from Th2-skewed cultures by positive selection using the MACS IL-4 Secretion Assay as specified in “Materials and methods.” The graph above shows the staining level for IL-4 in the selected cells (solid line), in the residual cells (filled histogram), and in the parental Th2 culture (dotted line) in a typical separation. RNA from IL-4⁺ and IL-4⁻ cells was extracted for quantitative real-time RT-PCR. GATA-3 transcript levels (expressed as percent of PPIA) are shown below and are the mean ± SEM of 4 experiments. *P = .05 relative to IL-4⁻. (C) Correlations (Spearman) of IL-4 transcript levels with levels of IFN-γ, CRTH2, GATA-3, and T-bet in Th1 and Th2 cultures and in CRTH2-sorted preparations. Transcript levels, relative to PPIA, were normalized across donors by conversion to percent of the average in parallel preparations. Each dot is the mean ± SEM of 4 donors.