Anti tgfβ pe

Cells required for intracellular cytokine staining were cultured for 6 h in RPMI 1640 containing PMA (Sigma-Aldrich) and ionomycin (Sigma-Aldrich). GolgiStop (BD Biosciences) was added to the culture for the last 4 h. Cells were surface stained with anti–CD19-PeCy7 or Pacific Blue (BioLegend), anti–TGF-β–PE (R&D Systems), anti-CD11c FITC or allophycocyanin (eBioscience), or anti-F4/80 allophycocyanin (eBioscience). Cells were then washed and stained with anti-mouse IFN-γ PE, anti-mouse IL-17 Alexa Fluor 647 (eBioscience), or anti-mouse IL-10 PE and analyzed by flow cytometry. Where possible, all gates were set using isotype control Abs. All samples were run on the LSRII or LSR Fortessa and analyzed using FlowJo software (Tree Star, Ashland, OR). All Abs were purchased from BD Biosciences unless otherwise stated.

Cells required for intracellular cytokine staining were cultured for 4–6 hr in RPMI containing PMA (Sigma-Aldrich) and ionomycin (Sigma-Aldrich). Golgi-Stop (BD Biosciences) was added to the culture for the last 4hr. Cells were surface stained with anti-CD19-AF700 or V450 (BioLegend, CA, USA), anti-TGFβ-PE (R&D Systems) and anti-CD11c FITC (eBioscience). Cells were then washed and stained with anti-mouse IL-6 FITC or v450 (eBioscience) and analyzed by flow cytometry. Where possible, all gates were set using isotype control antibodies or fluorescence minus one (FMO). All samples were run on the LSR Fortessa and analyzed using FlowJo software (Tree Star, OR, USA). IL-6 levels were assayed in supernatants using the IL-6 CBA Flex kit and buffer set (mouse and human; BD Biosciences) and analyzed using the FACS Verse. TGFβ1 was assayed by ELISA (R&D Systems) after sample acidification.