Pe cy7 anti cd25

Freshly isolated CD3⁺CD25⁻ murine T cells were stimulated by Dynabeads mouse T-activator CD3/CD28 (Gibco) according to the supplier’s protocol. 5 × 10⁴ BM- or FL-MSCs seeded in 6-well plates 1 day before were then co-cultured with 25 × 10⁵ activated CD3⁺CD25⁻ murine responder T cells (MSC to T cell ratio used 1:5) in a total volume of 3 ml of 50% RPMI-50% MEMα media. 1 × 10⁵ activated and non-activated murine CD3⁺CD25⁻ T cells grown alone in 50% RPMI-50% MEMα medium were used as controls. After either 1 or 3 days, murine T cells were harvested by gentle aspiration and stained with either VIOBLUE-anti-CD4, VioBright FITC-anti-CD8α, PE-anti-GITR, PE-Cy7-anti-CD25 (all from Miltenyi Biotec) and PE-Cy5.5-anti-Foxp3 (eBioscience) or anti-CD4-VIOBLUE, FITC-anti-CD8α, PE-Vio770-anti-ICOS, APC-anti-TNFR2 (all from Miltenyi Biotec), and PE-Cy5.5-anti-Foxp3 (eBioscience) antibodies (Abs).

Freshly isolated CD3⁺CD25⁻ murine T cells were stimulated by Dynabeads mouse T-activator CD3/CD28 (Gibco) according to the supplier’s protocol. 5 × 10⁴ BM- or FL-MSCs seeded in 6-well plates 1 day before were then co-cultured with increasing numbers of activated murine CD3⁺CD25⁻ responder T cells (MSC to T cell ratios used 1:1, 1:2, 1:4, 1: 6, 1:8, and 1:10) in a total volume of 3 ml 50% RPMI-50% MEMα medium. 1 × 10⁵ murine activated and non-activated CD3⁺CD25⁻ murine T cells grown alone in culture were used as controls. After 4 days, T cells are harvested and stained using the following Abs: VIOBLUE-anti-CD4, FITC-anti-CD8α, PE-Cy7-anti-CD25, PE-anti-CTLA4, APC-anti-TNFR2 (all from Miltenyi) and Foxp3-PE-Cy5.5 (eBioscience) or anti-CD4-VIOBLUE, FITC-anti-CD8α, PE-anti-GITR, PE-Vio770-anti-ICOS (all from Miltenyi Biotec), and PE-Cy5.5-anti-Foxp3 (eBioscience).