Il 13 elisa

Bone marrow cells (5 × 10⁵ cells per well) were plated in a round bottom 96-well plate and co-cultured with IL-2, IL-7, IL-33 at 10 ng/ml (Biolegend), and recombinant parasite-derived proteins, at 37°C, 5% CO₂ for 5 days. Supernatants were collected and analysed by IL-5, IL-6 and IL-13 ELISA according to manufacturer’s instructions (Thermo Fisher). Where biotinylated constructs were used to assess binding to bone marrow ILC2s, BALB/c or ST2-deficient bone marrow cells were cultured for 5 days in IL-2, IL-7 and IL-25 at 10 ng/ml (Biolegend) to support ILC2 proliferation, prior to incubation for 20 min at 4°C with HES and recombinant proteins, followed by biotinylated recombinant proteins for a further 20 min, as indicated, and avidin-PE to detect binding.

Following manufacturer’s instructions, mouse IL-5 and IL-13 ELISA (ThermoFisher Scientific) were utilized to assess cytokines secretion levels. Moreover, 32-plex (MCYTMAG70KPX32) and 41-plex (HCYTMAG-60K-PX41) Millipore Human Cytokine panel kits were used for assessment of additional cytokines, following manufacture specific protocols for multiplexed fluorescent bead-based immunoassay detection (MILLIPLEX^® MAP system, Millipore Corporation, Missouri, USA). The curve was completed using different concentrations of the cytokine standards each time, and then evaluated using MasterPlex2012 software (Hitachi Solutions America, Ltd.), as previously described before^{32 (link),46 (link)}.

Samples were evaluated using IL-17A ELISA (432501; BioLegend), IL-13 ELISA (88-7137-88; Thermo Fisher), IL-4 ELISA (431104; BioLegend), IL-5 ELISA (431204; BioLegend), IFN-γ ELISA (430801; BioLegend), TNF-α ELISA (430904; BioLegend), and GM-CSF ELISA (432201; BioLegend) kits according to the manufacturers’ instructions. Briefly, microwell plates (Corning Costar; 9018) were coated with capture antibodies overnight at 4 °C and blocked with ELISA diluent 1× for 1 h at room temperature. Samples and twofold serially diluted standards were incubated at room temperature for 2 h, and then detection antibodies and streptavidin-HRP were added. Then, a 1× TMB solution and a stop solution (2 N H₂SO₄) were added. The optical density was analyzed at 450 nm. Between all steps, the plates were washed at least 3 times with wash buffer (1× PBS, 0.05% Tween-20).