Mouse il 1β elisa max deluxe set

Cells in complete medium in 24-well plates (5 × 10⁵ cells/1 mL/well) were incubated overnight at 37°C/5% CO₂. The medium was removed and the cells incubated for 4 h with 1 μg/mL LPS in complete medium (1 mL/well). Cells were then washed three times with physiological medium (147 mM NaCl, 2 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, and 10 mM HEPES; pH 7.4). Cells were finally preincubated for 30 min in the absence or presence of compound (as indicated) in physiological medium (0.5 mL/well) and then incubated for 20 min in the absence or presence of ATP (as indicated). The samples were centrifuged (11,000 ×g for 30 s) and the cell-free supernatants were stored at −80°C until required. IL-1β concentration was measured using the Mouse IL-1β ELISA MAX™ Deluxe Set (BioLegend, San Diego, USA) according to the manufacturer's instructions. Lactate dehydrogenase (LDH) activity in cell-free supernatants and cell lysates was measured using the Cytotoxicity Detection Kit^PLUS (Roche, Mannheim, Germany) according to the manufacturer's instructions. LDH release is presented as a percentage of maximal LDH release.

The levels of IL-1β (Cat # 432604, Mouse IL-1β ELISA MAX™ Deluxe Set; BioLegend, London, UK) and IFNγ (Cat # 430804, Mouse IFN-γ ELISA MAX™ Deluxe Set; BioLegend) in the supernatant of the eosinophils-M1 macrophages were assessed using enzyme-linked immunosorbent assay (ELISA) as per the guidelines provided by the manufacturer.