Mice were sacrificed using an overdose of ketamine (160 mg/Kg) and xylazine (25 mg/Kg) at 2 weeks from initiation of the challenge (O2, IR, or O2+IR). Bronchoalveolar lavage (BAL) was then performed through a 20-gauge angiocatheter (BD Pharmingen, San Diego, CA), with the intra-tracheal instillation of 1 ml of phosphate-buffered saline (PBS) containing an anti-protease cocktail (Sigma) and 5 mM EDTA given in 0.5 ml increments [13 (link),14 (link),36 (link)]. An aliquot was immediately separated to measure total leukocyte cell counts (cells/ml BAL fluid) using a Coulter Cell and Particle Counter (Beckman Coulter, Miami, FL). The remaining lavage fluid was centrifuged at 1,200 rpm for 10 min and the cellfree supernatant was frozen at −80 °C for cytokine and protein analysis.
The amount of total protein in the BAL fluid was assayed using the BCA Protein Assay Kit (Pierce, Rockford, IL) as per manufacturer's instructions. Absorbance was read at 560 nm (MRX Microplate Reader, Dynatech Laboratories, Chantilly, VA) and protein levels in mg/ml of BAL fluid were calculated. The results are reported as fold change from control at week 0.
The amount of total protein in the BAL fluid was assayed using the BCA Protein Assay Kit (Pierce, Rockford, IL) as per manufacturer's instructions. Absorbance was read at 560 nm (MRX Microplate Reader, Dynatech Laboratories, Chantilly, VA) and protein levels in mg/ml of BAL fluid were calculated. The results are reported as fold change from control at week 0.
