Upon termination, the mice were anesthetized by subcutaneous injection of Hypnorm/Dormicum mixture diluted in sterile water 1:2 (Hypnorm: fentanyl citrate 0.75 mg/kg and fluanisone 23.8 mg/kg, VetaPharma Leeds, UK; Dormicum: midazolam 11.9 mg/kg, Roche a/s, Hvidovre, Denmark). Heart blood was withdrawn. Lungs were flushed twice with sterile 0.9% NaCl through the trachea to obtain BAL fluid, the used volume was calculated as 1 ml 0.9% NaCl/25g mouse weight and varied from 0.7 to 0.9 ml. BAL fluid was stored on ice until centrifugation at 400g for 10 min at 4°C. The BAL cells were re-suspended in 100 µl medium (HAM F-12 with 1% penicillin/streptomycin and 10% fetal bovine serum). Acellular BAL fluid was recovered and stored at −80°C. The total number of living and dead cells in BAL was determined by NucleoCounter NC-200TM (Chemometec, Denmark) from diluted cell suspensions following manufacturer's protocol. The total cell counts were calculated for each mouse.
Samples for COMET assay were prepared from 40 µl re-suspended BAL cells and 60 µl freezing media (HAM F-12, 1% penicillin/streptomycin, 15% fetal bovine serum and 10% DMSO). Samples were divided into two aliquots and immediately frozen at −80°C. The rest of the cell re-suspension (40 µl) was used to estimate the number of granulocytes (neutrophil and eosinophil), macrophages, lymphocytes, and epithelial cells in BAL fluid. The cell suspension was centrifuged at 55g for 4 min in Cytofuge 2 (StatSpin, TRIOLAB, Brøndby, Denmark) and fixed for 5 min in 96% ethanol. All slides were stained with May-Günewald-Giemsa stain, randomized, and blinded before counting 200 cells/sample under light microscope with 100 × magnification (using immersion oil). The numbers of counted cells were expressed as % observations based on cell distribution of the 200 counted cells multiplied with the total number of cells for each mouse. Lung and liver tissue were divided into 4 parts, snap frozen in liquid nitrogen, and stored at −80°C.
Samples for COMET assay were prepared from 40 µl re-suspended BAL cells and 60 µl freezing media (HAM F-12, 1% penicillin/streptomycin, 15% fetal bovine serum and 10% DMSO). Samples were divided into two aliquots and immediately frozen at −80°C. The rest of the cell re-suspension (40 µl) was used to estimate the number of granulocytes (neutrophil and eosinophil), macrophages, lymphocytes, and epithelial cells in BAL fluid. The cell suspension was centrifuged at 55g for 4 min in Cytofuge 2 (StatSpin, TRIOLAB, Brøndby, Denmark) and fixed for 5 min in 96% ethanol. All slides were stained with May-Günewald-Giemsa stain, randomized, and blinded before counting 200 cells/sample under light microscope with 100 × magnification (using immersion oil). The numbers of counted cells were expressed as % observations based on cell distribution of the 200 counted cells multiplied with the total number of cells for each mouse. Lung and liver tissue were divided into 4 parts, snap frozen in liquid nitrogen, and stored at −80°C.
