Purified rat anti mouse cd16 32

For B cell staining, PerCP-Cy5.5-conjugated CD19 (BD Pharmingen) and PE-conjugated anti-IL-10 (BD Pharmingen) mAbs were used. For identification of T cells, PerCP-Cy5.5-conjugated CD4 (BD Pharmingen), PE-conjugated CD25 (Miltenyi Biotech, Auburn, CA), Alexa Fluor 488-conjugated anti-IFN-γ (BD Pharmingen), PE-conjugated anti-IL-17A (BD Pharmingen), and Alexa Fluor 647-conjugated anti-Foxp3 (BD Pharmingen) were used. Cells from purified B cells of three different groups or the cocultured cells were stimulated with a leukocyte activation cocktail (BD Pharmingen, San Jose, CA, USA) for 5 h, followed by blocking with purified rat anti-mouse CD16/32 (BD Pharmingen) for 10 mins at 4°C. For surface staining, cells were stained with CD19-PerCP-Cy5.5, CD-25-PE, or CD4-PerCP-Cy5.5 mAbs. Cells were washed, fixed, permeabilized, and stained for detection of intracellular cytokines or transcription factors with IL-10-PE, IFN-γ-Alexa Fluor 488, IL-17A-PE, or Foxp3-Alexa Fluor 647 mAbs. Cells were subsequently analyzed using a FACSCanto II flow cytometer (BD Biosciences, Franklin Lakes, NJ). Dead cells and crystalline silica particles were gated out according to forward scattering (FSC) and side scattering (SSC). Cells were analyzed with FlowJo Software (TreeStar).