Ifn γ apc clone xmg1.2

Intracellular staining was performed as previously described.²⁴ (link) For this study, the following antibodies were used for surface staining: LIVE/DEAD Fixable Violet Dead Cell stain kit (Invitrogen), CD19 (V450; clone 1D3; BD Biosciences), CD4 (FITC; clone RM4-5; BD Biosciences), CD8 (APC-Cy7; clone 53–6.7; Abcam). For intracellular staining the following antibodies were used: IFN-γ (APC; clone XMG1.2; Biolegend), TNF-α (PE; clone MP6-XT22; ebioscience), IL-2 (PeCy7; clone JES6-SH4; ebioscience), CD3 (PerCP/Cy5.5; clone 145-2C11; Biolegend). All data were collected using a LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star, Ashland, OR) and SPICE v5.2 (free available from http://exon.niaid.nih.gov/spice/). Boolean gating was performed using FlowJo software to examine the polyfunctionality of the T cells from vaccinated animals. For flow cytometry, cells were gated on singlets using SSC-H by SSC-A followed by gating on LIVE-DEAD (dump channel), CD3⁺ CD4⁺ CD8⁻ T and CD3⁺ CD8⁺ CD4⁻ T cells to examine the CD4⁺ and CD8⁺ T-cell populations secreting IFN-γ, TNF-α, and IL-2 cytokines. 1 × 10⁶/wells cells were stimulated with H1HA consensus pooled peptides for 5 hours and 500,000 events were collected by the LSRII.