Dexamethasone does not completely suppress tumor-specific T cell activities in antigen-naïve mice.

Healthy mice received activated pmel-1 T cells labeled with the congenic marker Thy1.1 on day −2, followed by daily administration of dexamethasone (3 mg/kg) from day −2 to day 6. On day 0, mice were vaccinated with gp100, and spleens were harvested for flow cytometry analysis on day 7. The cells were stimulated with 10 ng/ml phorbol 12-myristate 13-acetate PMA, Sigma‑Aldrich) and 1 µg/ml ionomycin (Sigma‑Aldrich) in the presence of 10 µg/ml brefeldin A (Sigma‑Aldrich) at 37°C for 4 h. The cells were stained with Zombie Yellow, FITC-conjugated anti-CD3, APC/Cyanine7-conjugated anti-CD8 and Pacific Blue-conjugated anti-CD45, PE/Dazzle594-conjugated anti-B220, anti-F4/80 and anti-Ly6G, followed by fixation, permeabilization and staining with APC-conjugated anti-Granzyme B using Intracellular Staining Fixation Buffer and Intracellular Staining Permeabilization Wash Buffer (10X) according to the manufacturer's protocols.