Apc conjugated anti tnf

Cultured T cells were incubated with antigens (including BKV‐OPPs, BKV peptide matrices, minimisation peptide and defined peptide epitopes) in R10 containing GolgiPlug (BD Pharmingen, San Diego, CA) alone for IFN‐γ analysis, and with GolgiStop (monensin; BD Pharmingen) and anti‐CD107a antibody for multiparametric analysis. After 4 h of incubation at 37°C and 6.5% CO₂, the cells were washed with PBS containing 2% FBS (wash buffer) and the pellet was resuspended in wash buffer containing FITC‐conjugated anti‐CD4 and PerCP‐Cy5.5‐conjugated anti‐CD8 antibodies for IFN‐γ analysis, or with PerCP‐Cy5.5‐conjugated anti‐CD8 and PE‐Cy7‐conjugated anti‐CD4 for multiparametric analysis, and then incubated at 4°C for 30 min. Cells were then washed twice with PBS, fixed and permeabilised with Cytofix/Cytoperm solution (BD Pharmingen) for 20 min. Cells were then washed and incubated with PE‐anti‐IFN‐γ for IFN‐γ analysis or with PE‐conjugated anti‐IL‐2, Alexa Fluor 700‐conjugated anti‐IFN‐γ and APC‐conjugated anti‐TNF for multiparametric analysis (BD Pharmingen, San Diego) at 4°C for 30 min. Stained cells were washed twice with Perm/Wash buffer, resuspended in PBS containing 1% paraformaldehyde and acquired using a BD LSRFortessa. Post‐acquisition analysis was conducted using FlowJo software (TreeStar; FlowJo LLC, Ashland, OR).