Bronchoalveolar Lavage Neutrophil Quantification

The detailed procedures were described previously [28 (link), 29 (link)]. The mice were intubated with a 24-gauge cannula after their tracheas were surgically isolated. The lungs were flushed with 1 ml PBS back and forth for 3 times. Protein concentration in the BAL was determined by a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA). BAL cell smear was made using cytospin (Shandon, Pittsburgh, PA, USA). The slides were visualized using Hema3 staining (Fisher Scientific, Middletown, VA, USA). Neutrophils and monocytes were identified by a certified laboratory technologist in a blinded fashion. BAL cells were also used for flow cytometry analysis after lysing erythrocytes.