Tnfα fitc

Briefly, cells were incubated for 6 h at 37°C in 5% CO₂ with pools of overlapping 15-mer long peptides at a final concentration of 2 μM each, stimulated with SEB (Sigma) at 1 μg/ml or left unstimulated (negative control). The secretion inhibitor brefeldin A (Sigma) was added for the final 5 h of culture. After the 6 h incubation, surface staining for CD3-PercP, CD4-PECy7, CD8-PB (unless otherwise stated, staining reagents, instrumentation and software were from BD Biosciences) and live/dead fixable aqua dye (Life Technologies) was performed and then followed by intracellular staining for IFNγ-APC, TNFα-FITC, MIP-1β-PE, CD154-APC-eFluor780 (eBiosciences, San Diego, CA). Analysis was done on a two-laser CANTO II flow cytometer (BD Biosciences) and data analyzed with FlowJo software (TreeStar Inc., Ashland, OR). Spectral compensation was performed for each experiment with each individual mAb used in the surface and intracellular cytokines staining using compensation particles (BD Biosciences).

Splenocytes or PBMCs isolated from whole blood were stimulated with peptide at 2 μg/ml, ionomycin and phorbol myristate acetate (PMA) at 2.0 μg/ml and 0.5 μg/ml, respectively, or tissue culture media with 1% DMSO as a negative control. The cultures were supplemented with anti-CD107a PE-conjugated mAb (eBioscience). The cells were incubated at 37 ^oC, 5% CO₂ for 2 hours prior to the addition of Brefeldin A and monensin (BD Biosciences) and then left in culture overnight. The cells were centrifuged briefly, washed in PBS plus 5% BSA (Sigma-Aldrich) and the pellet re-suspended in 40 μl of CD16/32 with LIVE/DEAD fixable aqua stain (Molecular Probes, Invitrogen). Cells were washed, a mastermix of anti-membrane marker mAbs was prepared containing CD4 APC/Cy7 (Biolegend), CD3 PerCP-eFluor710 and CD8a eFluor 450 (both from eBioscience) and 40 μl added to each tube. The cells were incubated at 4 ^oC for 30 min and then permeabilized using Fix/Perm solution (Becton-Dickinson) for 20 min at 4 ^oC. The cells were washed with Perm Wash buffer (Becton Dickinson) and a mastermix of anti-intracellular molecule mAbs was prepared containing IFN-γ PE-Cy7, IL-2 APC and TNF-α FITC (all from eBioscience). The cells were incubated at 4 ^oC for 30 min, washed and resuspended in Perm Wash buffer prior to running on an LSRII flow cytometer (Becton-Dickinson).