Anti p stat3 antibody

PBMCs from healthy individuals were prepared and stained with CD14 and TNFRI or TNFRII (R&D Systems). For intracellular cytokine staining, cells were incubated for 5 h with PMA (50 ng/mL, Sigma-Aldrich) and ionomycin (0.5 μg/mL, Sigma-Aldrich) or with lipopolysaccharides (LPS, 100 ng/mL, Sigma-Aldrich) in the presence of GolgiPlug (BD Biosciences). The resulting cells were first surface-stained with anti-human CD4 antibody (BD Biosciences) or CD14 antibody (eBioscience) and then stained with antibodies to human IFNγ (BD Biosciences), IL-17, IL-6, or IL-1β (eBioscience) by using Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences). For p-STAT3 staining, cells were preactivated by anti-CD3/CD28 (1 μg/mL) for 24 h, fixed with 2% paraformaldehyde for 10 min at 37°C, and permeabilized by adding 100% pre-cold methanol for 30 min on ice. The resulting cells were stained with anti-human CD4 antibody, anti-p-STAT3 antibody (BD Biosciences), or isotype control (BD Biosciences).

RPMI-1640, RF10 (RPMI-1640 + 20 mM HEPES), DMEM high-glucose, and fetal bovine serum (FBS) were purchased from Hi-Media (Mumbai, India). Anti-mouse biotin-conjugated antibodies (lineage cocktail–biotin, and Thy1.2-biotin), anti-mouse fluorescence conjugated antibodies (CD4-FITC, CD8-PE, CD44-FITC, CD25-PE, c-Kit- PE/cy5.5 MHCII-FITC, and CD11c-PE), purified anti-mouse antibodies (CD4, CD8, CD45, Ki67, STAT3, IKAROS, IRF8, IL-10, and IL-10R), and CytoFix/CytoPerm solutions were procured from BD-Pharmingen or Biolegend (San Diego, CA, USA). Anti-pSTAT3 antibody and rmIL-10 were purchased from BD Biosciences (San Jose, CA). Aminoethylcarbazole (AEC) chromogen solution, and aqueous mounting media were procured from VECTOR Laboratories Inc. (Burlingame, CA).