Anti thy1.2 percp

The immunodominant RNEU_420-429 (PDSLRDLSVF) peptide and LCMV NP_118-126 (RPQASGVYM) negative control peptide were synthesized at >95% purity in the Johns Hopkins Biosynthesis and Sequencing Facility. The anti-OX40 antibody was produced from the OX86 hybridoma, a gift from the Weinberg lab. The hybridoma was grown in protein-free hybridoma media (Gibco) and purified over a protein G column (BD Pharmingen). Purified rat IgG was used as an irrelevant control (Jackson Laboratories). Antibodies for flow cytometry were: anti-DR5-PE, anti-CD24-APC, anti-AnnexinV-Pacific Blue, anti-Thy1.2-PerCP, anti-Thy1.2-Pacific Blue, anti-CD8-FITC, anti-Bcl-2-APC (Biolegend); anti-FasL-PerCP-efluor710 (eBioscience); anti-AnnexinV-PE, anti-Thy1.2-FITC, anti-IFNγ-PE, anti-TNFα-APC, and purified anti-Fas (BD Pharmingen); anti-Survivin-PE (Cell Signaling Technology); purified anti-CD24 and polyclonal rabbit IgG (Santa Cruz).

RNEU_420–429 (PDSLRDLSVF) and the negative control peptide LCMV NP_118–126 (RPQASGVYM) were produced in the Johns Hopkins Biosynthesis and Sequence Facility at a purity >95%. Antibodies used for flow cytometry studies were: anti-CD8-FITC (BD Biosciences), anti-CD8-PE (BD Biosciences), anti-CD8-PErCP (BD Biosciences), anti-CD8-APC (BD Biosciences), anti-galectin-3-AF647 (Biolegend), anti-galectin-3-PE (R&D), anti-PD1-PE (eBioscience), anti-LAG-3-PE (eBioscience), anti-Thy1.2-PerCP (Biolegend), anti-CD44-Pacific Blue (eBioscience), anti-CD11b-PE (BD Biosciences), anti-CD11c-FITC (BD Biosciences), anti-B220-APC (BD Biosciences), anti-Ly6C-PerCP-Cy5.5 (eBioscience), anti-IFNγ–PE (BD Biosciences), anti-IFNγ-Pacific Blue (eBioscience), and anti-Granzyme B-APC (BD Biosciences). Cellular division was assessed by labeling of high-avidity neu-specific CD8⁺ T cells with 1.5 μM CellTrace CFSE cell proliferation kit (Invitrogen) prior to adoptive transfer (8 (link)). Permeability was assessed with LIVE/DEAD Fixable Aqua Dead Cell Stain (Invitrogen). Antibody staining was conducted at 4°C for 20 minutes in FACS buffer (PBS, 5%FBS, 0.02% NaAzide).