Fitc conjugated anti mouse ly 6g gr 1 antibody

To obtain BAL cells from mice, lungs were lavaged three times with 0.4 ml of cold PBS. Total cell counts in the BAL were counted via a hemocytometer. BAL was further labeled with FITC-conjugated anti-mouse Ly-6G (Gr-1) antibody (Thermo Fisher Scientific) to determine the percentage of neutrophil via flow cytometry. Neutrophil counts were calculated by multiplying the percentage of neutrophil by the total number of total cells in the BAL. BAL was then centrifuged at 800g for 5 min to collect supernatant for analysis of total protein and cytokine levels. Protein concentration in BAL was determined using a BCA Protein Assay Kit. Cytokine levels were analyzed via ELISA for IL-1β (ThermoFisher Scientific) and IL-10 (R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol. OD values of the plates were detected by using SpectraMax 190 Microplate Reader (Molecular Devices, San Jose, CA).

To obtain BAL cells from mice, the tracheas were canulated and the lungs were lavaged three times with 0.5 ml of cold PBS. Total white blood cell counts in the BAL were directly examined using a hemocytometer. BAL was then labeled with FITC-conjugated anti-mouse Ly-6G (Gr-1) antibody (Thermo Fisher Scientific, Waltham, MA) and analyzed for the percentage of neutrophils via flow cytometry. Neutrophil counts in the BAL were calculated by multiplying the percentage of neutrophils by the total number of white blood cells. BAL samples were then centrifuged at 400 x g for 5 min at 4°C to collect supernatant. Protein concentration in BAL supernatant, which is an index of capillary leakage during lung inflammation, was determined using a BCA Protein Assay Kit (Thermo Fisher Scientific). Cytokine levels of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ in the BAL were examined by ELISA (R&D Systems, Minneapolis, MN) per manufacturer’s protocol.