Kc elisa kit

BALF analyses were performed on Day 1 after intratracheal administration of bleomycin as previously described (22) . Briefly, the lungs and trachea were exposed and a 20-gauge intravenous catheter was inserted into the trachea. A total of 1 mL of PBS was instilled three times and withdrawn from the lungs via an intratracheal cannula. More than 95% of the fluid was recovered as BALF, which was then centrifuged at 1,000 rpm for 5 min at 4°C. The supernatant was collected and stored at -80°C for use in ELISA. The levels of AIF-1, IL-6, TNF-α, and KC in the BALF were measured using commercial ELISA kits (an AIF-1 ELISA kit; USCN Lifescience, an IL-6 ELISA kit and a TNF-α ELISA kit; eBioscience, an KC ELISA kit; R&D Systems), according to the manufacturer's instructions. The optical density was measured at 450 nm using a SoftMaxPro40 plate reader. Each measurement was determined in three separate experiments on BALF. The total number of cells in BALF was counted using a Fuchs-Rosenthal hemocytometer (ERMA Inc., Tokyo, Japan). The BALF solution was placed in a cytospin (Cytospin 2; Shandon Inc., Pittsburgh, PA, USA), centrifuged at 700 rpm for 10 minutes, and stained with Diff-Quick (Sysmex, Kobe, Japan). The number of total cells, neutrophils, and macrophages were counted. At least 200 cells per slide were evaluated on the basis of morphological criteria using a light microscope.