Bms613

Serum and BALF were diluted with PBS, and lung samples were prepared by homogenization in PBS-containing protease inhibitors (Complete, Roche Diagnostics). Commercially available ELISA kits were used to quantify IFN-γ (BMS606, eBioscience), IL-4 (BMS613, eBioscience), IL-10 (BMS614/2, eBioscience), IL-17A (BMS6001, eBioscience), and C5a (ELM-CCC5A-1, Ray biotech) in BALF, serum, and lung tissues, according to the manufacturers’ instructions.

The lungs were lavaged through a tracheal cannula with 0.8 ml of PBS three times at 24 h after the last aerosol challenge. The BALF was collected and centrifuged at 1,500×rpm for 5 min at 4°C to pellet leukocytes, whose number was determined by using hemocytometer. The leukocytes were then subjected to cytospin preparation and stained with Diff-Quick (Sysmex, Kobe, Japan). At least 200 leukocytes on each slide were subjected to differential counting of macrophages, neutrophils, lymphocytes and eosinophils according to the standard morphological criteria. Concentrations of IL-4, IL-5, IL-13 and IFN-γ in the BALF were determined with the ELISA kits, BMS613, BMS610, BMS6015 and BMS606, respectively (eBioscience, San Diego, CA).

The cell supernatant was collected and, in a first set of experiments, levels of a large set of cytokines were measured using the MILLIPLEX MAP Mouse Cytokine/Chemokine Multiplex Assay (Merck; MCYTOMAG-70K). Subsequent experiments focused on the subset of cytokines whose levels were increased by LPS (namely IL-1β, TNF-α, and IL-6). Their levels were measured using a commercially available simple plex 5th generation cartridge (Bio-Techne, Minneapolis, MN, USA; SPCKC-MP-005977) and the readings were taken using Ella automated immunoassay system (Bio-Techne) according to the manufacturer’s instructions. In addition, IL-4 and IL-10 were also measured using ELISA (catalog BSM614 and BMS613, respectively, from Thermo Fisher Scientific) and the multimodal plate reader Ensight (PerkinElmer).