Pe cy7 tnfα

Cryopreserved PBMCs were thawed and 1 × 10⁶ PBMC were plated per well of a 96-well plate in a total volume of 200 μL of R10 medium (RPMI-1640 medium supplemented with 10% fetal bovine serum, L-glutamine, penicillin, and streptomycin) along with anti-CD28, anti-CD49d, and fluorescein isothiocyanate-conjugated anti-CD107a antibodies (BD Biosciences) and 1 μg/mL/peptide of the relevant peptide pool. R10 containing 0.5% DMSO was used as a negative control, and a mixture of 50 ng/mL phorbol 12-myristate 13-acetate and 1 μg/mL ionomycin was used as a positive control. Cells were incubated at 37 °C for one hour prior to addition of brefeldin A and monensin (BD Biosciences) and then left to continue incubating overnight. The next day, cells were washed and stained with LIVE/DEAD Aqua (Invitrogen, Life Technologies, Thermo Fisher Scientific) followed by the surface antibodies Brilliant Violet 785-conjugated anti-CD3 (BV785-CD3), BV605-CD4, BV650-CD8 (BioLegend), Alexa700-CD14, and Alexa700-CD19 (BD Biosciences). After fixation in 4% formaldehyde, cells were permeabilized and stained with the antibodies eFluor450-IFN-γ, PE-Cy7-TNFα, PE-MIP-1β, APC-IL-2, and PE-Cy5-CD154 (BD Biosciences). Data were collected using a BD LSRFortessa flow cytometer (BD Biosciences).