Anti mouse cd8 apc cy7

Single-cell suspensions were generated from the indicated organs followed by Lysis of RBCs with ACK solution. Following Fc Receptor blocking with anti-CD16/32 (2.4G2), cells were stained with the following antibodies obtained from BD Bioscience or Biolegend: anti-mouse CD8-APC-Cy7 (53–6.7), anti-mouse CD8-PECy7 (53.6–7), anti-mouse CD4-PECy7 (RM4–5), anti-mouse CD3-APC (145.2C11), anti-mouse CD3-Pacific Blue (17A2), anti-mouse CD19-PE (6D5); anti-mouse CD45.1-PECy7 (A20); anti-mouse CD45.2-APC (104); anti-mouse CD11c–APC (N418), anti-mouse CD11b–BV421 (M1/70). FACS analyses were performed on a FACS Calibur or a FACS Canto II (BD Biosciences). Data was analyzed with FlowJo software (Treestar).

Single-cell suspension from spleens of immunized animals (BALB/c mice 35 d.p.i or C57BL/6 mice 28 d.p.i) were prepared as described before and stimulated with 5 μg/mL of peptide pools that span both the lumazine synthase, GT8 or HA domains, or individual Trp2 (SVYDFFVWL) and Gp100₂₅ peptide (EGPRNQDWL) (GenScript) 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. ICS gate for CD8⁺IFNγ⁺ population is set as in Figure 1C and Supplementary Figure S4.