B-1 cells were identified by flow cytometry as live, CD3/4/8− F4/80−, GR-1−, CD19hi IgM-a+ IgD-alo CD43+ CD5+/− cells. A total of 2 mg AF6-78.2.5 antibody was given

for six weeks by bi-weekly injections, after which time allotype chimeras HM781-36B were maintained for at least two additional months before conducting experiments. To generate B-2-derived plasma cells, BALB/c mice were infected with influenza A/Mem/71 for 10 days as described previously 27. For reconstitution of RAG-1−/− mice, mice were irradiated with 850 rd full body γ-irradiation and reconstituted 16 h later with 2×106 total BM, or BM depleted of IgM+ cells via magnetic cell depletion using an auto-MACS (Miltenyi Biotec, Auburn, CA, USA). Mice were bled 6 weeks after reconstitution for analysis of serum IgM levels. Single-cell suspensions from spleen, peritoneal cavity wash out, BM and peripheral (pooled inguinal and axillares) LNs of individual mice were cultured in the absence of further stimuli in complete RPMI 1640 media (RPMI 1640, 2 mM L-glutamine, 100 μg/mL of penicillin and streptomycin, 10% heat-inactivated

fetal calf serum, and 50 μM 2-ME) at 37°C, 5% CO2 to assess spontaneous IgM secretion. Supernatants Carfilzomib were harvested after 16–18 h and analyzed by ELISA for presence of total and influenza virus-binding IgM. Total and virus-specific IgM secreting antibody-forming cells were enumerated by ELISPOT as previously described 56. B-1 and B-2 cell-derived IgM antibody-producing foci (AFC) were determined using Ig-allotype-specific monoclonal antibodies. Briefly, 5 μg/mL of anti-IgM (331, Demeclocycline not allotype-specific) or 1000 HAU/mL of purified A/Mem/71 were coated onto 96-well plates (Multi-Screen HA Filtration, Millipore, Bedford, MA, USA). After plates were blocked (PBS with 4% bovine serum albumin (BSA)), 2-fold serially diluted single-cell suspensions from various tissues were prepared and incubated overnight in complete RPMI 1640 media at 37°C, 5% CO2 chamber. Binding was revealed with in-house biotinylated allotype-specific anti-IgM (DS-1.1 for IgMa and AF6-78.2.5 for IgMb) followed by SA-HRP (Vector Labs, Burlingame,

CA, USA). Spots were developed with 3-amino-9-ethylcarbazole (Sigma Aldrich, St. Louis, MO, USA) and counted with the help of a stereomicroscope. Data are expressed as the number of IgM-secreting AFC per input cells. IgM production was quantified by sandwich ELISA as described previously 56. Briefly, 5 μg/mL of anti-IgM (331) antibody was coated onto 96-well plates (Maxisorb, Nalgene Nunc, Rochester, NY, USA). After blocking non-specific protein binding, serially diluted culture media was added to plates. Binding was revealed with biotinylated anti-IgM antibodies. The levels of total IgM production (μg/ml) were calculated using purified IgM as the standard. Single-cell suspensions from peritoneal cavity wash outs (PerC), spleen and BM were stained with the following antibody conjugates at predetermined optimal concentrations.