Murine Splenocyte Activation Assay

Single-cell suspension from spleens of immunized C57BL/6 mice were prepared as described before and stimulated with peptides that either span the lumazine synthase domain, or individual Trp2₁₈₈ (SVYDFFVWL), Gp100₂₅ (EGPRNQDWL) and Tyrp1₄₅₅ (CTAPDNLGYM) peptides at 5 µg/mL for 5 hours at 37°C in the presence of 1:500 protein transport inhibitor (ThermoFisher) and anti-mouse CD107a-FITC (ThermoFisher). The cells were then incubated with live/dead Fixable Violet Dead Cell Stain Kit (for 405 nm excitation) for 10 minutes at room temperature, and surface stained (anti-mouse CD4-BV510, Biolegend, Catalog: 100559; anti-mouse CD8–APC-Cy7, Biolegend, Catalog: 100714) at room temperature for 30 minutes. The cells were then fixed and permeabilized according to manufacturer’s instructions for BD Cytoperm Cytofix kit and stained with anti-mouse IL2–PE-Cy7 (BioLegend, Catalog: 503832), anti-mouse IFNγ-APC (BioLegend, Catalog: 505810), anti-mouse CD3e–PE-Cy5 (BioLegend, Catalog: 100310), and anti-mouse TNFα-BV605 (BioLegend, Catalog: 506329) at 4°C for 1 hour. The cells were subsequently analyzed with LSR II 18-color flow cytometer. The data was analyzed with FlowJo V10.6.1.