Fitc mouse anti human ifn γ

PBMCs were seeded into a 10-mL sterile tube, containing 1 mL of RPMI media (Gibco) containing penicillin/streptomycin 10.000 U/10 mg (Sigma-Aldrich), 10% fecal bovine serum (FBS) (Gibco), at a final concentration of 5 × 10⁵ cell mL⁻¹. To stain intracellular cytokines, cells were activated with 10 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich), and 1.25 pM calcium ionophore (Sigma-Aldrich) during 2 h at 37 °C in a CO₂ incubator (Heraeus 6000). Then, Brefeldin A was added to the cell solution, to the final concentration of 5 μg·mL⁻¹ (Sigma-Aldrich) and incubated overnight at 37 °C, in a CO₂ incubator [7 , 21 (link)].
Cells were fixed and permeabilized with Immunostep Fix and Perm Kit, adding solution A (fixative) and incubated for 15 min at room temperature. Then, cells were washed with Facs Flow buffer (BD) and centrifugated at room temperature during 7 min at 1700 rpm in the Heraeus Labfuge 400. Solution B (permeabilization) was added to the cells, with conjugated intracellular antibody cocktail (FITC Mouse Anti Human IFN-γ (BD); PE Mouse Anti Human TNF-α (BD Pharmigen); PerCP-Cy5 Mouse Anti Human CD3 (BD); Anti IL-17A-APC Human (Miltenyi Biotec); at room temperature during 30 min in the dark). Cells were washed with Facs Flow buffer (BD) and resuspended in 1 mL Facs Flow buffer (BD). Cells were screened with Facs Calibur (BD), and analyzed with FlowJo software v8.7.

After three days of stimulation the CD3/CD28 beads were removed from the cells, and the cells were re-stimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) in the presence of monensin (3 µM) as previously described [59] . The cells were then stained with PerCP/Cy5.5 anti-human CD4 (317428, BioLegend), fixed and permeabilized with BD Cytofix/Cytoperm followed by BD Perm/Wash according to the manufacturer instructions, and finally stained intracellularly with FITC mouse anti-human IFN-γ (554551, BD Pharmingen), anti-human IL-17A APC (17–7179, eBioscience), FITC Rat Anti-Human IL-4 (554484, BD Pharmingen) or PE anti-human IL-13 (501903, BioLegend). Data were acquired on a FACSCalibur (BD, Brøndby, Denmark) with CellQuest Pro software, and subsequently analyzed using FlowJo software.