T-cell proliferation/Treg-suppression assay

For mouse Treg-suppression assays, CD4⁺CD25^hi Tregs in tumor cell suspensions were sorted into KLRG1⁺ or KLRG1^- cells and cultured at 1:2, 1:4, and 1:8 ratios with Carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled CD4⁺CD25^- cells (0.25×10⁵) isolated from the spleen of the same mouse. T cell–depleted, mitomycin C–treated splenocytes (0.25×10⁵) were added as antigen presenting cells (APCs) and cultures were stimulated with soluble anti-CD3 (clone 2C11, ebioscience) at 0.5 µg/ml concentration for 3 days.