Tnp ova

To generate a sufficient source of basophils for the assay, we administered IL-3 complexes (10 µg IL-3 [R&D Systems] complexed to 50 µg anti–IL-3 [MP2-8F8; BioLegend] for 5 min at room temperature) to Basoph8 mice 3 d before migration assays (Finkelman et al., 1993 (link)). Mice were then sensitized with 5 µg monoclonal IgE against TNP (C38-2; BD) 24 h before the assay. On the day of the migration assay, splenocytes were enumerated, and a total of 12,500 basophils were placed in the upper chamber of the transwell apparatus with LTB4 at 5 nM ± 100 ng/ml TNP-OVA (Biosearch Technologies) placed in the lower chamber analogous to published protocols (Ansel et al., 1999 (link)). For transendothelial migration assays, 1.5 × 10⁵ bEnd.3 cells (an immortalized, mouse brain endothelial cell line) were placed on gelatinized transwell inserts 40 h before the assay (Montesano et al., 1990 (link)). Basophils in the lower chamber were assessed by flow cytometry after a 4-h incubation at 37°C. Each condition was performed in duplicate. Inhibitors PF 431396 and PF 573228 (Tocris Bioscience) were diluted to the indicated concentration and placed in both the upper and lower wells of the transwell just before addition of the basophils.

To determine antigen-specific IgM or IgG titers, 20 µg/ml TNP-OVA (Biosearch Technologies) was used for coating (4°C overnight). For measurement of total serum IgG concentrations, 1 µg/ml isotype-specific coating antibodies was used (IgG, IgG1, IgG2a, IgG2b, and IgG3; SouthernBiotech). The coating was performed at 4°C overnight. For the anti-dsDNA ELISA, the plates were coated with 5 µg/ml double-stranded calf thymus genomic DNA (Sigma-Aldrich) for 2 h at room temperature. For detection, 1 µg/ml goat anti–mouse IgM-HRPO (SouthernBiotech) or goat anti–mouse IgG-HRPO (Thermo Fisher Scientific) and as a substrate TMB (Enzo Life Sciences) were used.