Biotinylated goat anti mouse igm

Splenic cell suspensions were treated with ACK lysis buffer to remove red blood cells and then stained with biotinylated anti-CD43 (ebioR2/60; eBioscience), anti-CD1d (1B1), anti-CD11b (M1/70; BioLegend), and anti-CD11c (N418; BioLegend) antibodies by standard procedures. To obtain IgM⁻ B cells, biotinylated goat anti–mouse IgM (SouthernBiotech) was also included. Magnetic Dynabeads M-280 Streptavidin (Thermo Fisher Scientific) were used to remove labeled cells according to the manufacturer’s instructions.

For ELISpot analysis of total IgM⁺ and IgG⁺ ASCs, Multi-Screen filter plates (Millipore) were activated with 35% ethanol, washed with PBS and coated with either anti-IgM (SouthernBiotech, no. 1020-01) or anti-IgG (SouthernBiotech, no. 1030-01) in PBS. Single bone marrow cell suspensions were prepared as above and cultured at 250,000 cells ml^–1 in RPMI 1640 medium (Invitrogen) supplemented with FBS (10% v/v, Invitrogen), penicillin/streptomycin/amphotericin B (1% v/v) and 50 μM β-mercaptoethanol (RPMI-FBS) at 37 °C for 16 h. Following removal of supernatants, plates were incubated with either biotinylated goat anti-mouse IgM (SouthernBiotech, no. 1020-08) or goat anti-mouse IgG1 antibiotic (SouthernBiotech, no. 1070-08) for 2 h and, after washing, incubated with horseradish peroxidase-conjugated streptavidin. Plates were developed using the Vectastain AEC peroxidase substrate kit (Vector Laboratories). The stained area in each well was quantified using CTL ImmunoSpot software (Cellular Technology), and is depicted as the number of spots for quantification

ELISA plates (Nalge Nunc, Rochester, NY) were coated with 500 ng/well of NP25 conjugated with BSA (Biosearch Technologies). To detect IgM, IgG1, IgG3 or IgA, plates were coated with unconjugated goat anti‐mouse IgM (Southern Biotech, , Birmingham, AL), IgG (Southern Biotech), or IgA (BD, Franklin Lakes, NJ), respectively. After incubation overnight (4°C), washing with PBS + 0.05% Tween20 and blocking for 1 h with PBS containing 2% dry milk, 50 μL of culture supernatant was added in a total volume of 150 μL, followed by threefold serial dilutions in blocking buffer and incubated for 2 h at room temperature (RT). Plates were washed six times, and primary antibodies, biotinylated goat anti‐mouse IgM, HRP‐coupled anti‐IgG1, HRP‐coupled anti‐IgG3 (Southern Biotech, Birmingham, AL), or biotinylated goat anti‐mouse IgA (BD Franklin Lakes, NJ), were added in 100 μL PBS/well followed by incubation for 1.5 h, at RT. After six washes, streptavidin‐HRP was added to biotinylated antibodies in 100 μL PBS/well and incubated for 1 h at RT. The assay was developed with TMB substrate (KPL), the reaction was stopped with 1 m H₂SO₄, and the OD was read at 450 nm using an Asys Expert 96 ELISA reader (Biochrom Ltd, Cambridge, UK). For detection of soluble TACI, the mouse TACI/TNFRSF13B DuoSet ELISA kit (R&D Systems) was used, according to the manufacturer's instructions.