T-cell Proliferation Assay Using CFSE

T-cell proliferation was measured by carboxyfluorescein succinimidyl ester (CFSE) dye dilution as reported previously.³⁹ (link) In brief, LN cells from B6 mice were first labeled with CSFE dye and stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin at concentrations of 50 ng/mL and 500 μM, respectively, and then were cultured with G-MDSCs or MDSC subsets at different ratios for 5 days at 37°C with 5% CO₂ in complete RPMI 1640 medium (Life Technologies) supplemented with 100 U/mL penicillin, 100 U/mL streptomycin, 292 μg/mL l-glutamine, and 10% fetal bovine serum (Life Technologies). In some experiments, N-acetylcysteine (1 mM; Sigma, St. Louis, MO) or anti-mouse PD-L1 antibody (10 μg/mL; BioLegend) was added to LN cell culture. Cells were harvested and stained with CD4 and CD8 antibodies and analyzed for CFSE dye dilution using a BD LSRFortessa flow cytometer.