BALF and Serum Analysis in COPD Mouse Model

BALF and serum analysis was conducted as described in our previous study [26 (link)]. The animal study was conducted twice to verify reproducibility. In the first study, three mice were used for the evaluation of BALF and serum, with the remaining four mice used for other studies, such as the morphological analysis (H&E stain and Masson's trichrome stain) and the expression of specific proteins related to the occurrence of COPD (IHC). BALF collection was scheduled from all mice after anesthetization with 50 mg/kg Zoletil (Virbac, Carros, France), and the tracheas were cannulated with disposable animal feeding needles. Lavage was performed with three 0.4 mL aliquots of cold phosphate-buffered saline (PBS), and BALF samples were collected and immediately centrifuged at 3,000 rpm for 5 min (Sorvall Legend Micro 17R, Thermo Fisher Scientific Inc., Waltham, MA, USA). The cell pellets were resuspended in PBS, and total and differential cell counts were obtained. The number of total cells and differential cells was counted with a Hemavet Multispecies Hematology System (Drew Scientific Inc., Waterbury, CT, USA). After cell collection (if used), the animals were sacrificed by an additional Zoletil injection. IgE levels in the serum were measured using a specific mouse IgE ELISA kit (BD Bioscience, catalog number 555248, San Jose, CA, USA) in accordance with the manufacturer's protocols.