Anti ifn γ alexa 647

Peripheral blood mononuclear cells (PBMCs) or splenocytes were plated in 96-well round-bottom plates and stimulated by the addition of pools of overlapping 20-mers covering the whole protein at a final concentration of 2 μg/ml in the presence of 1 μg/ml BD GolgiPlug and incubated for 6 h (37°C; 5% CO₂). After cell surface labeling with anti-CD4-e450 and anti-CD8-peridinin chlorophyll protein (PerCP)/Cy5.5 antibodies (Affymetrix eBioscience), as well as a LIVE/DEAD fixable aqua dead cell stain kit (Thermo Fisher Scientific), the cells were fixed with neutral buffered formalin solution containing 4% formaldehyde (Sigma-Aldrich) for 5 min at 4°C. Then, intracellular staining was performed with anti-tumor necrosis factor (TNF)-Alexa 488, anti-interleukin 2 (IL-2)-phycoerythrin (PE), and anti-IFN-γ–Alexa 647 antibodies (Affymetrix eBioscience) diluted in BD Perm/Wash buffer. Flow cytometry data were analyzed using a BD LSR II flow cytometer with BD FACSDIVA (Becton, Dickinson) and FlowJo (Tree Star) software. Antigen-specific cells were identified by gating on size, double-negative live cells, and either CD4⁺ or CD8⁺ surface expression. Background responses in unstimulated wells were subtracted from responses of stimulated T cells before statistical analysis in Prism 6.07 (GraphPad) (see Fig. S7 in the supplemental material).

PBMC or splenocytes were plated in 96-well round bottom plates and stimulated by the addition of individual Pb9 peptide or pools of overlapping 20mers covering the whole protein (TRAP or Ii as indicated) at a final concentration of 2 µg/mL in the presence of 1 µg/mL BD GolgiPlug™, and incubated for 6 hours (37 °C, 5% CO₂). After cell surface labelling with anti-CD4-e450 and anti-CD8-PerCP/Cy5.5 antibodies (Affymetrix eBioscience) as well as LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific), cells were fixed with neutral buffered formalin solution containing 4% formaldehyde (Sigma Aldrich) for 5 minutes at 4 °C. Then, intracellular staining was performed with anti-TNF-Alexa488, anti-IL-2-PE and anti-IFN-γ-Alexa647 antibodies (Affymetrix eBioscience) diluted in BD Perm/Wash buffer. Flow cytometry data was analysed using a BD LSR II Flow Cytometer with BD FACSDIVA (Becton Dickinson) and FlowJo (Tree Star) software. Antigen-specific cells were identified by gating based on size, doublet negative, live cells and either CD4⁺ or CD8⁺ surface expression. Background responses in unstimulated wells were subtracted from responses of stimulated T cells before statistical analysis in Prism 6.07 (GraphPad).