Alexa fluor 488 conjugated anti ifn γ

Fluorescein (FITC)-conjugated anti-CD25 (catalog number 555431), phycoerythrin (PE)-conjugated anti-CD132 (catalog number 555900), allophycocyanin (APC), peridinin-chlorophyll proteins (PerCP)- and Pacific Blue (PB)-conjugated anti-CD4 (catalog numbers 555349, 347324 and 558116, respectively), PerCP- or FITC-conjugated anti-CD8 (catalog numbers 347314 and 555634, respectively), Alexa Fluor 647-conjugated anti-CD127 (catalog number 558598), FITC-conjugated anti-CD45RA (catalog number 555488), PE-conjugated anti-phosphorylated STAT5 (612567), PE Cy7-conjugated anti-PD-1 (catalog number 561272), Alexa Fluor 488-conjugated anti-IFN-γ (catalog number 557718) and Fixable Viability 510 (564406) were purchased from BD Biosciences. PE-conjugated anti-Bcl-2 (MHBCL04) was purchased from Thermo Fisher Scientific. Cell samples were acquired on a FACS Aria II flow cytometer (BD, USA) and analyzed with FlowJo software (Tree Star, San Carlos, CA, USA). Lymphocytes were gated based on their forward scattering and side scattering parameters, followed by the use of forward scatter area vs. forward scatter height dot-plot for doublet discrimination. The subsequent analyses were performed on viable cells (FV510^—) (S1A Fig)

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).