Anti mip1β apc

HLA‐B3501‐NY9‐specific CD8⁺ T‐cell clones were stimulated with 2 or 20 μm of NY9 peptide and were incubated for 7 h in the presence of GolgiPlug (BD Biosciences), GolgiStop (BD Biosciences) and anti‐CD107a‐AF488‐FITC (BD Biosciences). Following stimulation, cells were surface stained for 30 min with anti‐CD8‐PerCP‐Cy5.5 (BD Biosciences), anti‐CD4‐BV650 (BD Biosciences) and Live/Dead Fixable Near‐IR Dead Cell Stain (Life Technologies). Cells were fixed and permeabilized using BD Cytofix/Cytoperm solution (BD Biosciences) and then intracellularly stained with anti‐IFN‐γ‐BV421 (BD Biosciences) as well as anti‐TNF‐PeCy7, anti‐MIP1β‐APC and interleukin‐2‐PE (all BD Biosciences) for a further 30 min. Cells were acquired on a CytoFLEX Flow Cytometer. Post‐acquisition analysis was performed using FlowJo software (version 10.7.1; BD Biosciences). Cytokine detection levels identified in the no‐peptide control condition were subtracted from the corresponding test conditions in all summary graphs to account for nonspecific, spontaneous cytokine production.

CD8⁺ T cell lines were stimulated with 1 μM of the cognate or the homologous peptide and were incubated for 4–5 h in the presence of GolgiPlug, GolgiStop and anti-CD107a-FITC (dilution 1:100) (all BD Biosciences). After stimulation, cells were surface stained for 30 min with anti-CD3-BV480 (1:100), anti-CD8-PerCP-Cy5.5 (1:50) and anti-CD4-BV650 (1:100) antibodies (all BD Biosciences) and Live/Dead Fixable Near-IR Dead Cell Stain (Life Technologies). Cells were fixed and permeabilized using BD Cytofix/Cytoperm solution (BD Biosciences) and then intracellularly stained with anti-IFN-γ-BV421 (1:100), anti-TNF-PE-Cy7 (1:100), anti-IL2-PE (1:100) and anti-MIP-1β-APC (1:100) antibodies (all BD Biosciences) for a further 30 min. Cells were acquired on the BD FACSymphony A3 system using the FACSDiva software (v.9.0.). Post-acquisition analysis was performed using FlowJo software (v.10). Cytokine detection levels identified in the no-peptide control condition were subtracted from the corresponding test conditions in all summary graphs to account for non-specific, spontaneous cytokine production.