Anti mouse tnfα clone mp6 xt22

Zap70(AS) CTL were cultured with or without hamster anti-CD3ε (145-2C11, Becton Dickinson Bioscience, United Kingdom) for 5 hr at 37°C in 96-well round bottom plates at approx 0.5–2 × 10⁶ cells/well in c-RPMI medium containing 5 μg/ml GolgiPlug (Becton Dickinson) ±10 μM 3-MB-PP1. The cells were then washed with PBS (containing 0.1% BSA and 0.02% sodium azide), stained with anti-mouse CD8α-PerCPCy5.5 (Pharmingen) for 30 min on ice, permeabilised by paraformaldehyde fixation using the BD Cytofix/Cytoperm Kit (Becton Dickinson) and stained for intracellular cytokine production using anti-mouse IFNγ-FITC (clone XMG1.2), anti-mouse TNFα (clone MP6-XT22), and anti-mouse IL-2 (clone JES6-5H4) (Pharmingen, United Kingdom). Lymphocytes were washed and analysed on a FACScalibur and analysed using CellQuestPro software (Becton Dickinson). In each assay, any cytokine positive cells isolated from wells with no were subtracted from the % cytokine positive cells incubated with peptide to yield the final value.

Tumor lysates were prepared by mincing the tumor in DMEM (Sigma) and incubating in collagenase (Roche) at 37°C for 1 h. After washing in PBS, the cells were filtered through 70‐μm filters (BD Biosciences). 5 × 10 cells were re‐suspended in HBSS (Hank's balanced salt solution, Lonza) supplemented with 0.5% BSA (Sigma). Staining was performed at 4°C for 20 min, with the following antibodies: anti‐mouse CD45 (clone 30‐F11, Biolegend San Diego, CA, USA), anti‐mouse F4/80 (clone BM8, Biolegend San Diego, CA, USA), anti‐mouse cd11c (clone N418, Biolegend San Diego, CA), anti‐mouse I‐A/I‐E (clone M5/114.15.2, Biolegend San Diego, CA, USA); anti‐mouse TNFα (clone MP6‐XT22, BD Pharmingen), anti‐mouse Ly6C (clone HK1.4, eBioscience; San Diego, CA, USA); anti‐mouse NOS2 (clone 6/iNOS/NOS Type II, BD biosciences, San Diego, CA, USA) and mouse IgG2a K (BD biosciences, San Diego, CA, USA). For intracellular staining, Cytofix/Cytoperm and Permwash staining kit (BD Pharmingen) were used. Cells were detected using the BD FACS Canto II cytofluorimeter and analyzed with FlowJo software.