Inflammatory Markers and Cell Counts in Murine BAL

After anesthetizing the mice with xylazine and Zoletil 50^® on day 8, BAL fluid sampling and tracheostomy were performed as previously described [32 (link)]. ROS level was determined using 2′, 7′-dichlorofluorescin diacetate (DCF-DA, Sigma-Aldrich, Carlsbad, CA, USA), according to previously reported protocol [33 (link)]. Additionally, TNF-α, IL-6, and MCP-1 levels were determined using standard ELISA kits (BD Biosciences, San Jose, CA, USA), according to the manufacturer’s protocols. To calculate the number of inflammatory cells in BAL fluid samples, the samples were centrifuged using a CytoSpin 3 cytocentrifuge (Thermo Fisher Scientific, Waltham, MA, USA). Thereafter, the centrifuged preparations were stained on a slide using Diff-Quik^® reagent (SYSMEX, Kobe, Japan) and the inflammatory cells were counted using a light microscope at 400× magnification. The number of inflammatory celsl was determined as the average of counted cells in five different fields [16 (link)].