Following PFT measurements, bronchoalveolar lavage (BAL) was performed with three serial instillations of 0.8 mL of sterile PBS through the tracheostomy. Blood samples were obtained through IVC puncture and collected in EDTA tubes. BAL samples were centrifuged at 800×g at 4 °C for 10 min while plasma was separated by centrifugation at 2000×g for 20 min. The BAL and plasma supernatant were flash frozen in liquid nitrogen and saved at − 80 °C for further analysis.
The BAL cell pellet was suspended in 100 μL sterile PBS and 1 mL of RBC lysis solution (Miltenyi Biotec, Auburn, CA). The sample was incubated for 10 min at room temperature and then centrifuged at 300×g for 5 min. The supernatant was discarded and the cells were resuspended in 100 μL sterile PBS and placed on a cytospin slide. Total and differential counts of BAL fluid (BALF) were determined using the May-Grunwald-Giemsa stain (300 cells per animal) as previously described7 (link). Counts were performed in biological duplicates.
The BAL cell pellet was suspended in 100 μL sterile PBS and 1 mL of RBC lysis solution (Miltenyi Biotec, Auburn, CA). The sample was incubated for 10 min at room temperature and then centrifuged at 300×g for 5 min. The supernatant was discarded and the cells were resuspended in 100 μL sterile PBS and placed on a cytospin slide. Total and differential counts of BAL fluid (BALF) were determined using the May-Grunwald-Giemsa stain (300 cells per animal) as previously described7 (link). Counts were performed in biological duplicates.
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