Apc cy7 conjugated anti ifnγ clone 4s b3

To measure intracellular IFNγ and IL-17A production by T-cell subsets, cells were either left untreated or stimulated with PMA (50 ng/ml) plus ionomycin (1 μg/ml) in the presence of Brefeldin A (10 μg/ml) in RPMI medium containing 10% FCS for 6 h, as previously described [13 (link)]. Activated PBMCs were surface stained using FITC-conjugated anti-CXCR3 (clone G025H7), PE-conjugated anti-CCR6 (clone G034E3), APC-conjugated anti-CCR4 (clone L291H4) and PE-Cy7-conjugated anti-CD4 (clone RPA-T4) mAbs, fixed and then permeabilized using commercially available Perm Wash Buffer (BioLegend, San Diego, USA). IFNγ and IL-17A were detected using APC-Cy7-conjugated anti-IFNγ (clone 4S.B3) and PerCP-conjugated anti-IL-17A (clone BL168) mAbs, respectively (obtained by BioLegend, San Diego, USA).

Intracellular IFN-γ and IL-17 production by PBMCs was measured by flow cytometry utilizing anti-human fluorochrome-conjugated monoclonal antibodies. Briefly, detection of intracellular cytokine production involved use of peridinin chlorophyll protein (PerCP)–conjugated anti-IL-17A (clone BL168) and APC-Cy7-conjugated anti-IFN-γ (clone 4S.B3), obtained by BioLegend (San Diego, CA, USA). Following surface staining and fixation, cells were then permeabilized using Perm/Wash buffers (BD Biosciences). Incubation with optimally titrated antibodies followed. Online Resource 2 summarizes cell subset characterization by surface and intracellular marker staining.