Micro 12tm

To obtain the BALF, the tracheas of the anesthetized mice were exposed and cut just below the larynx. A flexible polyurethane tube (outer diameter; 0.4 mm, length; 4 cm; BD Biosciences, San Jose, CA, USA) attached to a blunt 24-gauge needle (Boin Medical Co., Seoul, Korea) was placed inside the trachea. Next, the lungs were lavaged once with 800 μL of sterile cooling phosphate-buffered saline (PBS). The BALF samples were centrifuged for 5 min at 1500 ×g 4°C. The supernatants were moved to new microcentrifuge tube and frozen at -70°C; the remaining cell pellets were resuspended in 100 μL of PBS. Total cells were counted using a hematocytometer. To observe the differential cell counts, the same number of BALF cells was centrifuged on the slides at 500 rpm for 5 min using a Cytospin apparatus (Micro 12TM, Hanil Co., Seoul, Korea). The slides were dried and stained with Diff-Quick solution (Sysmex Co., Kobe, Japan). At least 500 cells per slide were evaluated to analyze differential leukocyte counts.

After mice were anesthetized, the tracheas were exposed and cut just below the larynx. A flexible polyurethane tube attached to a blunt 24-gauge needle, with a 0.4-mm outer diameter and length of 4 cm (Boin Medical Co., Korea), 800 µL of cold PBS was inserted into the trachea. The BALF samples were collected and centrifuged for 5 min at 1500 rpm at 4°C. After centrifugation, the supernatants were collected and quickly frozen at −70°C. The cell pellets were resuspended in 100 µL of ACK hypotonic lysis solution (Sigma) and incubated for 2 min to lyse the RBCs. Next, 900 µL of PBS was added, and the cell suspension was centrifuged for 5 min at 3000 rpm at 4°C. The supernatants were then decanted, and the cell pellets were resuspended in 100 µL of PBS. After each procedure, the cells were centrifuged onto microscope slides for 5 min at 500 rpm using a Cytospin apparatus (Micro-12TM; Hanil Co., Korea). The microscope slides were air-dried and stained with Diff-Quik (Sysmex Co., Japan). Cells on the stained slides were counted in a blinded manner under a light microscope. At least 500 cells were counted per slide.