Yellow viability dye

Peripheral blood mononuclear cells (PBMC) were isolated using Ficoll (Fisher Scientific, Hudson, NH) and centrifugation. Where indicated, 5x10⁶ PBMCs were cultured for 3 days in the presence of anti-BCR (4ug/mL anti IgG Fab’2 fragment, Jackson Immunoresearch Laboratories, West Grove, PA), TLR9 agonist (CpG 2006;1ug/mL, Operon, Huntsville, AL) or TLR7/8 agonist (R848;1ug/mL, Invivogen, San Diego, CA). At day 3, PBMC were stained for B cell subset markers, yellow viability dye (Invitrogen, Carlsbad, CA) and CD86 FITC (clone 2331, BD Biosciences, San Jose, CA). Flow cytometric acquisition was performed on 30,000–50,000 CD19+ B cells as previously mentioned.

MDM and MoDC were characterized by flow cytometry. Briefly, the harvested cells were washed once with PBS containing 2% FBS. The cells were then incubated at room temperature with fluorochrome-conjugated monoclonal antibodies diluted in PBS/ 2% FBS for 20 minutes while protected from light. The cells were washed again, and the resulting cell pellet re-suspended in PBS/ 2% FBS. The sample data were acquired using BD LSR II Fortessa flow cytometer equipped with BD FACSDiva software. The antibody panel for MDM included: anti-CD11b (ICRF44)-Pacific Blue, anti-CD11c (N418)-AF700, anti-CD14 (M5E2)-FITC, anti-CD86 (2331 (FUN-1))-PE-Cy7, anti-CD163 (GHI61)-APC, anti-CD206 (15–2)-PE-Cy5, anti-HLA-DR (G46-4)-V500 and yellow VID live-dead stain. The MoDC panel included: anti-CD14 (M5E2)-PE, anti-CD40 (5C3)-FITC, anti-CD80 (L307.4)-Qdot605, anti-CD83 (HB15e)-PerCP-Cy5.5, anti-HLA-DR (G46-4)-V500 and yellow VID live-dead stain. Antibodies were purchased from either BD Biosciences or BioLegend. Yellow viability dye was purchased from Invitrogen. Data was analyzed using Community Cytobank data analysis software (Cytobank).