Mouse elisa max kit

PEMϕs were infected with the S. typhimurium SH100 strain as described above, and their culture supernatants were collected at the indicated time points. IL6 levels in PEMϕ supernatants were measured using the Mouse ELISA Max Kit (BioLegend) according to the manufacturer’s instructions.

To measure degranulation upon FcεRI ligation, CTMCs were incubated overnight with anti-TNP IgE at 0.5 μg/mL (clone IGELb4) in medium containing IL-3. The cells were washed twice with prewarmed Tyrode’s buffer (10 mM HEPES [pH 7.4], 130 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1.4 mM CaCl₂, 5.6 mM D-glucose, and 0.1% BSA) and stimulated with TNP₄-BSA in Tyrode’s buffer for the indicated periods. The culture supernatant was incubated with 4-MUAG (final concentration 0.5 mM) in citrate-phosphate buffer (pH 4.5; 52 mM citric acid, 94 mM Na₂HPO₄) for 15 minutes at 37°C, followed by the addition of Stop solution (2 M Na₂CO₃, 1.1 M glycine) to end the reaction. Fluorescence (excitation, 365 nm; emission, 450 nm) was measured on a microplate reader (VarioScan Flash, Thermo Fisher). The degranulation rate was calculated by dividing the β-hexosaminidase activity in the culture supernatant by that in the cell lysate, which was the supernatant of cells lysed with Tyrode’s buffer containing 0.5% Triton-X 100. Cytokine levels in the BMMC culture supernatants were measured using the Mouse ELISA Max Kit (BioLegend Mouse IL-6 ELISA MAX, 431302) or the Ready-Set-Go ELISA Kit (Thermo Fisher, 88-7324-88 for TNFα, 88-7137-88 for IL-13).