B cell phenotypic analysis protocol

For B cell phenotypic analysis PBMCs were stained similar to as previously described [28 (link), 29 (link)] with anti-CD19-APC-Cy7 (SJ25C1, BD), anti-CD20-AlexaFluor 700 (2H7, Biolegend, San Diego, CA), anti-CD3-PacificOrange (UCHT1, Invitrogen, Carlsbad, CA), anti-IgD-FITC (IA6-2, BD), anti-IgG-APC (G18-145, BD), anti-IgM-PE-Cy5 (G20-127, BD), anti-CD27-Qdot655 (CLB-27/1, Invitrogen), anti-CD21-V450 (B-ly4, BD), anti-CD14-PerCP-Cy5.5 (M5E2, BD), anti-CD138-biotin (B-A38, Abcam, Cambridge, MA), streptavidin Qdot800 (Invitrogen), anti-CD4-Qdot705 (S3.5, Invitrogen), anti-CD38-Qdot605 (HIT2, Invitrogen), anti-CD95-PE-Cy7 (DX2, Biolegend), anti-CD24-PE-AlexaFluor610 (SN3, Invitrogen), anti-CD183-PE (1C6/CXCR3, BD) and Live/Dead fixable aqua dead cell stain (Invitrogen). One-to-two million total events per sample were collected on an LSRII instrument (BD Biosciences). Staining and analysis performed in a blinded manner, linking sample with experimental group after gating was completed using FlowJo software (Treestar, Inc, Ashland, OR). Total PBMC were gated on lymphocytes using FSC and SSC. Live/Dead stain, anti-CD3, anti-CD4, and anti-CD14 were used to exclude dead cells, T cells, and monocytes respectively.