Wright s staining solution

The collected bronchoalveolar lavage fluid (BALF) was centrifuged at 1000×g for 15 minutes at 4°C, the supernatant was collected and frozen at -80°C for subsequent assays. The total number of inflammatory cells in the BALF was determined by counting cells with a blood cell counter (Beckman Coulter, Inc.) after resuspension of the cell precipitate in PBS and exclusion of dead cells by Tissue Blue staining. For analysis of cell numbers, 100 μl of BALF was centrifuged onto slides by Cytospin (Thermo Fisher Scientific, Waltham, USA). After the slides were dried, the cells were fixed and stained using Wright’s staining solution (32857, Sigma, USA) according to the manufacturer’s instructions. The number of neutrophils was sorted to determine the percentage of neutrophils by a laboratory technician who was unaware of the experimental design. The frozen BALF supernatant was thawed and mixed thoroughly and the total protein concentration was determined by the BCA (bicinchoninic acid) method.

Cellular morphology was assessed by light microscopy of Wright’s-stained cyto-smear preparations as follows: after treatment, cells were pelleted at 1000 rpm for 5 minutes and the supernatant discarded. Cyto-smear for microscopic examination was prepared by spreading a small drop of cell pellet on a glass microscope slide and air-drying. The smears were subjected to Wright’s staining and observed by microscope under high power field. Wright’s staining solution and hematoxylin-eosin staining solution were from Sigma-Aldrich.