Lung Epithelial Cell Stretch Assay

Immortalized mouse lung epithelial cells (MLE-15) were the generous gift of Jeffrey Whitsett, M.D. (Professor of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH) and were cultured as described previously.^{30 (link)} For cyclic stretch experiments, cells were plated on Pronectin coated Flexcell plates (Flexcell, Hillsborough, NC) and grown to confluence. Cells were then treated with a combination of 0.5 μg/ml Escherichia coli lipopolysaccharide (L2762, Sigma-Aldrich) and 100 units/ml of interferon gamma (315-05, Peprotech, Rocky Hill, NJ) or PBS control for 4 hours (Fig 1B). Cells were subsequently exposed to isoflurane (or control gas containing 23% oxygen, 5% CO₂, balance nitrogen) in an airtight chamber for 2 hours as described previously.^{31 (link)} Isoflurane, oxygen, and carbon dioxide concentrations were measured every 30 minutes as described previously.^{31 (link)} After isoflurane exposure, cell were incubated overnight prior to biaxial cyclic stretch (10% stretch, 0.5 Hz) delivered via a Flexcell Strain Unit FX-3000 (Flexcell) for 2 hours prior to isolating RNA or protein. Control cells were placed in the stretching device, but not subjected to stretch.