Thermo shandon cytospin 3

For protein and cellular analysis, the BAL components were separated by centrifugation. The protein concentration inside the BAL fluid was analyzed by a protein assay (Pierce^TM BCA Protein Assay, Fisher Scientific, Hampton, VA, United States) based on the manufacturer’s instructions. Albumin concentration in the BAL was measured via ELISA (Rat Albumin ELISA Kit, Bethyl Laboratories, Montgomery). The BAL samples were diluted 1:1000 and compared to the standard curve according to the manufacturer’s protocol. For cellular analysis, the BAL cells were separated from the rest of the BAL via centrifugation. Cytospin slides (Thermo Shandon Cytospin 3, Marshall Scientific, Hampton VA, United States) were prepared with 50.000 cells per BAL diluted in 100 μl phosphate buffer (PBS). Lymphocytes, macrophages and neutrophilic granulocytes were differentiated after staining the Cytospins with DiffQuik. Using Visiopharm^® Software a systematic uniform area sampling was performed and at least 100 to 200 cells were categorized and counted using a primary magnification of 40×. Furthermore, we assessed the concentration of IL-6 within the BAL using an ELISA Kit (DuoSet^® ELISA Rat IL-6, R&D Systems, Minneapolis, MI, United States). We utilized 96 well-plates and photometric measurements for cytokine concentration. Calculations were made with Magellan Software.

Prior to tracheostomy, mice were anesthetized by intraperitoneal injection of pentobarbital (50 mg/kg; Hanlim Pharma Co., Seoul, Korea). A 23-gauge needle was used to insert a silicone tube into the mouse trachea, which was connected to an 800-µl tuberculin injector to deliver 1 ml of Hank’s balanced salt solution (HBSS; Thermo Fisher Scientific, Waltham, MA, USA) to the lungs. The recovered bronchoalveolar lavage fluid (BALF) was centrifuged for 3 min at 10,000 rpm at 4 °C, and the supernatant was stored at − 70 °C. Whole cells were re-suspended in HBSS, and BALF cell smears were prepared using cytocentrifugation (Thermo Shandon Cytospin 3, Marshall Scientific, Hampton, NH, USA) and then stained with Diff-Quick (Sysmax, Kobe, Japan). The percentages of macrophages, eosinophils, lymphocytes, and neutrophils in BALF were determined by counting 500 leukocytes in randomly selected fields under a light microscope.