The expression of phenotypic Treg markers in coculture experiments by flow cytometry. PBMCs obtained from 5 healthy donors were cultured in the absence (PBMC) or presence of SK-N-SH (PBMC+SH), SK-N-AS (PBMC+AS), and SH-SY5Y (PBMC+SY5Y) for 4 days prior to flow cytometric analysis. (a) The sequential gating strategy of Treg enumeration. (b) The percentage of CD4+T cells of PBMCs. (c) The percentage of CD4+CD25+activated T cells of total CD4+T cells. (d) The percentage of CD4+CD25+Foxp3+Treg cell of total CD4+T cells. The results showed that the PBMC+SH coculture condition, but not those of PBMC+AS or PBMC+SY5Y, had significant increase in the Treg frequency as compared to the control condition (PBMC), suggesting that a particular microenvironment of SK-N-SH cells mediated Treg differentiation. Values represent mean±SD of 5 independent experiments (∗p<0.05; ∗∗p<0.001).