Intracellular Cytokine Staining Assay

ICS assay was performed according to a standard protocol, as described previously [17 (link)]. Briefly, splenocytes isolated from mice (2 × 10⁶ cells/well) were plated in 24-well plates and stimulated with a mix of VACV-specific peptides (SPYAAGYDL, SPGAAGYDL, VGPSNSPTF, KYGRLFNEI, GFIRSLQTI, and KYMWCYSQV) or with PMA (phorbol myristate acetate, 30 ng/mL) and ionomycin (1 µg/mL). Each peptide was added at a concentration of 20 µg/mL per well, and cells were incubated for 4 h at 37 °C in 5% CO₂, and for additional 16 h with Brefeldin A (5 μg/mL, BD Biosciences, Franklin Lakes, NJ, USA). On the next day, cells were stained with pre-titrated anti-CD3 MCA500SBB700 (Bio-Rad, Hercules, CA, USA), anti-CD8 FITC (BD Pharmingen, San Diego, CA, USA), and anti-CD4 PerCP (BD Pharmingen, San Diego, CA, USA), fixed, and permeabilized using Cytofix/Cytoperm solution (BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer’s instructions. Cells were then stained for intracellular cytokine detection with anti-IFN-γ APC (BD Pharmingen, San Diego, CA, USA). Samples were analyzed on a ZE5 flow cytometer (Bio-Rad, Hercules, CA, USA). Data were presented as the medians and ranges of variation.