Polyurethane flexible tube

To obtain BALF, the tracheas of anesthetized mice were exposed and cut immediately below the larynx. A polyurethane flexible tube (BD Biosciences, San Jose, California, USA) attached to a blunt 24-gauge needle was placed in the trachea, and the lung was lavaged once with 800 μL of sterile phosphate-buffered saline. BALF samples were centrifuged for 5 min at 3,000 rpm and 4 °C. The supernatants were decanted and immediately frozen at −70 °C. The cell pellets were resuspended and washed twice with phosphate-buffered saline. The total cell numbers were counted using a hemocytometer. To determine the differential cell counts, BALF cells were prepared using a cytospin apparatus and stained with Diff-Quik solution (Sysmex Co., Kobe, Japan) in accordance with conventional morphological criteria. At least 200 cells per slide were evaluated to determine the differential leukocyte counts.

To obtain BALF, the tracheas of anesthetized mice were exposed and cut just below the larynx. A polyurethane flexible tube (outer diameter: 0.4 mm, length: 4 cm; BD Biosciences, San Jose, California, USA) attached to a blunt 24-gauge needle was placed in the trachea, and the lung was lavaged once with 800 μl of sterile, warm PBS. BALF samples were centrifuged for 5 min at 1500 rpm at 4°C. Supernatants were then decanted and immediately frozen at −70°C. Cell pellets were resuspended and washed twice in PBS. Total cell numbers were counted using a hemocytometer. To determine the differential cells counts, BALF cell smears were prepared with a cytospin apparatus and stained with Diff-Quik solution (Sysmex Co., Kobe, Japan), in accordance with conventional morphological criteria. At least 500 cells per slide were evaluated to obtain differential leukocyte counts.