Murine elisa kit

Murine TNF-α, IL-6, IL-1b, IL-10, and TGF-β were measured in serum-free CM from WT-M0, WT-M2, TRIM59-CKO-M0, and TRIM59-CKO-M2 cells using murine ELISA kits purchased from eBioscience (San Diego, CA, USA) according to the manufacturer’s instructions. Murine IL-2 was measured in serum free CM from WT-M0, WT-M2, and TRIM59-CKO-M2 cells using murine ELISA kits purchased from R&D Systems (Minneapolis, MN, USA). Absorbance was measured at 450 nm and corrected at 540 nm using a microplate reader. Total protein concentrations in CM were calculated using CurveExpert 1.4. (CurveExpert and GraphExpert Software, Madison, AL). Statistical analysis was performed using Student’s t-test. A p < 0.05 was considered statistically significant.

Mice were anesthetized with an intraperitoneal injection of 1% pentobarbital sodium (50 mg/kg). Blood samples were collected from the retro-orbital sinus after the mice lost consciousness. Subsequently, blood samples were allowed to clot by leaving them undisturbed at 25 °C for 30 min. The clots were then removed to obtain serum via centrifugation at 1000 × g for 10 min at 4 °C. Part of the right lung from each mouse was homogenized with ELISA buffer and centrifuged to obtain lung tissue supernatants. Samples of murine serum, lung tissue supernatants, and cell culture supernatants were used to quantify the concentrations of IL-1β (R&D System, Minneapolis, MN, USA) and IL-18 (R&D System) by using murine ELISA kits, according to the manufacturer’s instructions. The MPO activity in the lung tissue was assessed by an MPO assay kit (R&D System).