Elisa max standard set mouse il 4

For ex vivo cell culture experiments in Fig. 3a–c, sort-purified pre-basophils and mature basophils (1 × 10⁴ cells) were separately cultured for 1 or 2 days in the absence of IL-3, followed by flow cytometric analysis. In Fig. 4e, f, mice were first intravenously administered with 300 μg of TNP-specific IgE antibody (IgE-Lb4) for IgE-sensitization to basophils, 1 day prior to basophil isolation. Sort-purified pre-basophils and mature basophils (2 × 10⁴ cells) were incubated with TNP-conjugated ovalbumin (TNP-OVA: 10 ng/mL) or control OVA (10 ng/mL) at 37 °C for 1 h or 6 h. In Fig. 4g, pre-basophils and mature basophils (2 × 10⁴ cells) were isolated from the bone marrow of non-sensitized mice and stimulated with the following reagents at 37 °C for 6 h: IL-3 (BioLegend: 10 ng/mL), IL-18 (BioLegend: 10 ng/mL), IL-33 (BioLegend: 10 ng/mL), LPS (Sigma: 100 ng/mL). The concentration of IL-4 in culture supernatants was measured by using ELISA MAX Standard Set Mouse IL-4 (BioLegend, catalog #: 431101).

After the splenic cells had been stimulated with RBD protein for 42 hours, the culture supernatant was harvested for the detection of mouse cytokines via ELISA (ELISA MAX™ standard Set Mouse IL-2, ELISA MAX™ standard Set Mouse IL-4, ELISA MAX™ standard Set Mouse IL-10, ELISA MAX™ standard Set Mouse IFN-γ, Biolegend). Due to the limited volume, harvested supernatant from one group was pooled, and three parallel wells were set. The following detection limits were obtained from the manufactures: IL-2 (detection limit 2.0 pg/mL), IL-4 (detection limit 2.0 pg/mL), IL-10 (detection limit 31.3 pg/mL), and IFN-γ (detection limit 15.6 pg/mL). Dilution factors of the samples were determined during a preliminary experiment. The experimental ELISA was carried out according to the manufacturer’s instructions.