Elisa kits for mouse tnf α

Wild-type C57BL/6 (WT-Stock no. 000664) and TLR4^−/− (Stock no. 029015) mice purchased from the Jackson Research Laboratories (Bar Harbor, ME, USA) and maintained in the pathogen-free vivarium. Eight to twelve-week-old mice were used for all the animal experiments, conducted following protocols approved by the Institutional Animal Care and Use Committee of The Ohio State University. Escherichia coli LPS Serotype O55:B5 S-form, dissolved in sterile saline was delivered through intranasal insufflation into anesthetized WT or TLR4^−/− mice (4 mg/kg in 25-μL saline). Control mice received an equal volume of saline. After 6 h, 3 days, and 15 days of LPS treatment, mice were euthanized with ketamine/xylazine, trachea was cut open, BALF was collected by instilling 1-mL sterile saline three times and aspirating. Aspirated BALF was centrifuged at 300 g to separate cells, and the supernatant was used for TNF-α, IL6, extravasated protein measurements, and EV isolation. Cytokine release was analyzed using R&D Systems ELISA kits for mouse TNF-α (catalog no. MTA00B) and IL-6 (catalog no. M6000B) following the protocols supplied by the manufacturer. Lung wet dry ratios and histological changes were determined as described previously [30 (link)].