Tbet apc

The cultured CFSE stained cells were incubated with Brefeldin A (Biolegend) concomitant with their restimulation. 6 hrs later the cells were harvested and stained. For T cell and B cell activation cells were harvested and stained as follow: with Aqua Live/Dead (Life Technologies) anti-human CD3 (PerCP5.5), CD4 (Alex700), CD19 (PE), IgG (BV421). The cell were fixed and permeabilized using BD Cytofix/Cytoperm kit according to the manufacturer’s protocol, after which intra-cellular staining was done with anti-human IFNγ (APC) (all fluorochrome-labeled antibodies from Biolegend). FACS analysis was gated on Live CD3+CD4+CFSE^low for proliferating T cells, or Live CD3-CD19+ CFSE^low for B cell proliferation. Cells were analyzed by FACS with BD LSRII. The following cell surface markers for T helper (Th) were used CxCR3 (BV421), CCR4 (APC), and CCR6 (PE). LIVE CD3+CD4+ cells were gated on CXCR3+CCR4–CCR6– cells (defined as Th1), CCR6+CCR4+CXCR3– (defined as Th17), CCR6+CXCR3+CCR4– (defined as Th1/Th17), and on CFSE^low for proliferation. The following fluorochrome-labeled antibodies were used for intra cellular staining for cytokines or transcription factors: IL-17A (BV421), IFNγ (APC), Tbet (APC), all from Biolegend, IL17F (PE, from eBioscience), RORγt (BD Pharmingen). FACS analysis was done with BD LSRII at our core facility.