Canto 2 4 blue 2 violet 2 red laser configuration

Six days after co-culture, CD8⁺ T cells were harvested and stained to assess CD107a expression following stimulation with PMA (25 ng/ml, Sigma-Aldrich) and ionomycin (1 µM, Sigma-Aldrich) for 3 hr. Cells were stained with anti-CD8-PerCP-Vio700, anti-CD45-RA-FITC, or isotype controls (Miltenyi Biotech, Paris, France). For membrane expression analysis, cells were then stained with anti-CD107a-PE or isotype control (BD Biosciences, San Jose, CA). For total expression analysis, cells were fixed, permeabilized using the IntraPrep Permeabilization Reagent Kit (Beckman Coulter), and stained with anti-CD107a-PE or isotype control. FACS data were acquired using a Canto II 4-Blue 2-Violet 2-Red laser configuration (BD Biosciences).

Four or 6 days after co-culture, CD8⁺/CD4⁺ T cells were harvested and manually counted before being processed for FACS analysis. Dead cells were excluded by using DAPI or the Zombie NIR fixable viability kit (BioLegend). Intracellular cytokines were assessed following stimulation with PMA (25 ng/ml, Sigma-Aldrich), ionomycin (1 µM, Sigma-Aldrich) for 4 hr, and brefeldin A (5 µg/ml, Sigma-Aldrich) for the last 3 hr. Cells were stained with anti-CD8-PerCP-Vio700 or anti-CD4-PerCP-Vio700, anti-CD45-RA-FITC, and then fixed, permeabilized using the IntraPrep Permeabilization Reagent Kit (Beckman Coulter) and stained with anti-Granzyme-APC, anti-TNF-α-PE, anti-IFN-γ-APC, anti-IL-17- PE-Cy7, anti-IL-10-PE or isotype controls (Miltenyi Biotech, Paris, France). The percentage of cell proliferation was estimated using Cell Trace Violet fluorescence loss. FACS data were acquired using a Canto II 4-Blue 2-Violet 2-Red laser configuration (BD Biosciences). Flow cytometry analyses were performed using Diva 8 (BD Biosciences). Human TNF-α concentration levels were quantified using ELISA following the manufacturer’s recommendations (BioTechne). Values below the detection limit were counted as zero.