Rat igg1 isotype control clone r3 34

Cell population and cytokine secretion of iNKT cells were measured by flow cytometry. Before staining cells, with a previously defined optimal dilution of monoclonal antibodies (Abs), the cells were pre-incubated with anti-CD16/32 (clone 93) to block non-specific FcRγ binding. The following Abs were used in this study: anti-CD3, anti-CD4, anti-CD8, and anti-CD49b (Biolegend, San Diego, CA, USA). For intracellular staining, BALF cells were incubated with brefeldin A (10 μg/ml) (BD Biosciences, San Diego, CA, USA) at 37°C for 1 hour then incubated with anti-CD16/32 Abs, followed by staining with PerCP/Cy5.5-conjugated CD3 and PE-conjugated PBS57 loaded CD1d tetramer (originally produced by the NIH tetramer facility, and supplied through Dr. David Serreze), permeabilized with Cytofix/Cytoperm reagent (BD Biosciences), and stained with Alexa Fluor® 488-conjugated anti-IFN-γ (clone XMG1.2), Alexa Fluor® 488-conjugated anti-IL-4 (clone 11B11), or rat IgG1 isotype control (clone R3-34) (BD Biosciences). Stained cells were assessed on a FACSCalibur (BD Biosciences) using FlowJo softwares (Tree Star, Inc., Ashland, OR, USA).