al was harvested for AECII according to our previously published protocol, or removed from the chest cavity for whole lung homogenization. AECII were isolated from lavaged lungs by dispase digestion followed by differential adherence on IgG plates. Homogenized lungs were, washed briefly in PBS and homogenized in 100 ml PBS/mg of tissue with protease inhibitors before the addition of Triton X-100 to 1%. 21560248 Homogenates were vortexed, and both AECII and lung homogenates were kept at 280uC until use. Prior to use, the total protein in all AECII and lung homogenate samples was quantified using the Bradford assay kit previously described for use in standardization of experimental analyses. Histology Whole lung specimens, from a subset of animals randomly chosen from each experimental group, were fixed in 4% paraformaldehyde at 2025 cm H2O MedChemExpress SAR 405 inflation pressure, embedded in paraffin and cut into 57 mm thick sections. Mouse lung sections were stained with Sirius Red/Fast Green FCF for collagen visualization. Morphological changes in 225 randomly chosen microscopic fields, spanning all experimental conditions at the chronic time point, were photographed with 20-fold magnification, and were quantified according to the numerical scale proposed by Ashcroft et al.. Visualization and quantification of CM-Dil labeled cells was achieved through counterstaining with 49,6-diamidino-2-phenylindole . In vitro adherent cells were fixed in 4% paraformaldehyde for staining. Overnight incubation with primary antibodies used for immunofluorescence included CCR2 and a-smooth muscle actin . Measurements of Lung Mechanics and Collagen Quantification A subset of animals randomly chosen from 
each experimental group were anesthetized with 7090 mg/kg pentobarbital sodium solution, tracheotomized, placed in a plethysmograph and connected to the Scireq small animal ventilator to measure pulmonary mechanics. Mice were mechanically ventilated at a rate of 150 breaths/min, tidal volume of 10 ml/kg, and positive end-expiratory pressure of 23 cmH2O. All maneuvers were computer controlled via Flexivent v5.2 software. Pressure-volume loops were generated by a sequential delivery of seven increments of air into the lungs from resting pressure to total lung capacity followed by seven expiratory steps during which air was incrementally released. Pressures at each of the incremental volumes delivered were recorded and graphed to give pressure-volume loops. The Salazar-Knowles equation was applied to measurements resulting from the pressure volume manipulations to calculate quasi-static compliance and hysteresis, which provides an estimate of the amount of airspace closure that existed before the PV loop maneuver. Negative pressure forced expirations were performed via rapidly switching the airway opening to negative pressure, resulting in the ability to measure Forced Vital Capacity. All measurements and maneuvers were performed in triplicate. Following pulmonary function testing, whole lungs were excised from each animal for total collagen content analysis via hydroxyproline assay kit according to the manufacturers 7906496 instructions. Briefly, whole lungs were washed in PBS, weighed and homogenized in 100 ml dH2O/mg of tissue. Tissue samples were then hydrolyzed at 120uC for 3 hours before transfer to a 96 well plate for oxidation of free hydroxyproline. Hydroxyproline content was then assessed by spectrophotometry at 570 nm. Proteomic Cytokine Analysis Cytokine levels were assessed in BAL a
