Cd43 negative selection

For ex vivo assays, naïve splenic B cells were purified by CD43 negative selection (Miltenyi Biotec), cultured at a density of 10⁶ per ml, and stimulated with either LPS (30 μg/ml, Sigma), LPS (30 μg/ml) + IL-4 (12.5ng/ml, R&D Systems), or LPS (10 μg/ml) + TGF-β (2ng/ml, R&D Systems) + anti-IgD dextran conjugates (300 ng/ml, Fina BioSolutions) for CSR to IgG3, IgG1, or IgA, respectively. For proliferation assays, naïve splenic B cells were labeled with 5 μM CellTrace Violet (CTV, Thermo Fisher Scientific) according to manufacturer’s protocol, activated with LPS, LPS+IL-4 or LPS+TGFβ+ anti-IgD dextran and dye dilution tracked from d0 to d4. Antibodies for flow cytometry were as follows: B220 (BV510, FITC, PerCPCy5.5; clone RA3–6B2), IgA (PE; clone mA-6E1), IgG1 (BV510, APC; clone X56), IgG3 (FITC; clone R40–82), CD69 (PE/Cy7; clone H1.2F3), CD86 (AF700; clone GL-1), MHC Class II I-Ab (eFLuor450; clone AF6–120.2), Fas (BV510; clone Jo2), GL7 (FITC, PerCP-eF710; clone GL7) and Zombie Red fixable viability dye; all were purchased from eBioscience, BD, and BioLegend. Samples were analyzed on an LSR II flow cytometer (BD). Cell sorting was carried out in a FACSAria cell sorter (BD). All data analysis was performed using FlowJo software (version 9.9; Tree Star).

OP9-GFP cells (12 (link)) were maintained
in MEMα (ThermoFischer) with 20% HI-FBS (Hyclone), glutamine,
β-mercaptoethanol, and penicillin/streptomycin. 1E4 sorted preproB cells
were added to 80–90% confluent OP9-GFP cells in 35 mm dishes in IMDM with
10% HI-FBS, 5 ng/ml murine FL, 1 ng/ml murine IL-7 (Peprotech), and
penicillin/streptomycin. Formation of mature myeloid and B lymphoid cells was
assessed using anti-CD11b-PE and anti-CD19-PerCP-Cy5.5 (1D3), and B cell
precursors were evaluated as done for marrow cells, in both cases gating on the
GFP^- population. Splenocytes were subjected to red blood cell
lysis and CD43-negative selection (Miltenyi). CD43^- cells were
cultured in RPMI with 15% HI-FBS, β-mercaptoethanol,
penicillin/streptomycin, and 20 ng/ml murine IL-4 (Peprotech) with either 10
μg/ml anti-murine CD40 (1C10, Biolegend) antibody or 25 μg/ml
E. coli O55:B5 LPS (Sigma), followed by analysis on day 4
using anti-B220-APC with anti-IgE-PE (RME-1, Biolegend) and anti-IgG1-BV421
(RMG1–1, Biolegend) or anti-IgG2b-PE (RMG2b-1, Biolegend). Serum obtained
by lancing the facial vein was analyzed for IgM, IgG, and IgA after 1:10,000
dilution using ELISA kits per the manufacturer’s instructions
(Invitrogen).