Cd86 fitc clone 2331

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.

Nine parameter flow cytometric analysis was performed on 200uL of whole blood. Lymphocytes were assessed by forward and side scatter, and stained for the following: anti-CD19-PECy5 (clone HIB19),anti- CD20- APC-H7 (clone L27), anti-CD10- APC (clone HI10a), anti-CD21- PE (clone B-ly4), anti- CD27- PECy7 (clone M-T271) (all from BD Biosciences, San Jose, CA) and anti-CD38- Alexa Fluor 700 (clone HIT2, Biolegend, San Diego, CA). At least 20,000 CD19+ B cells events were acquired on an LSRII cytometer driven by FACSDiVa version 6.2 software. Analysis was performed using FlowJo Version 10 software. BD TruCOUNT (BD Biosciences) beads were used for absolute counts of CD19+ B cells. For CD86 FITC (clone 2331, BD Biosciences, San Jose, CA) whole blood was stained for 10 minutes in the dark at room temperature, then lysed for 2 minutes with 2mL of BD Lyse (BD Pharmigen, San Diego, CA) and washed with 2mL of 0.1% BSA+ PBS, then fixed with 200uL of BD Stabilizing Fixative (BD Biosciences). For Ki-67(clone B56, BD Biosciences),whole blood was stained as above, but permeabilized with BD Cytofix/Permeabilization (BD Biosciences) on ice for 20 minutes, washed with 2mL of Perm Wash (BD Biosciences) and stained with 20uL of anti-Ki-67 FITC for 30 minutes on ice, washed twice with 2 mL of Perm Wash and fixed.