Alveolar Imaging and Cell Manipulation

All animal experiments were approved by the Institutional Animal Care and Use Committee of Columbia University Medical Center. We imaged isolated, blood-perfused lungs by laser scanning microscopy (LSM 510 META, Zeiss)^{11 (link)}. Alveoli were imaged to a depth of 40 μm from the pleura. We loaded alveolar cells with dyes and reagents by alveolar micropuncture^{11 (link)}. LPS concentrations were 1 mg (kg body weight)⁻¹ for all experiments, and 25 mg kg⁻¹ for survival studies. We infused calcein-stained S. aureus (1x10⁸ bacteria ml⁻¹) by alveolar micropuncture. For Ca²⁺ imaging (1 image 5s⁻¹), we microinfused alveoli with fluo-4. For photolytic Ca²⁺ uncaging^{17 (link)}, we targeted single cells, co-loaded with fluo-4 and the UV-sensitive Ca²⁺ cage, o-Nitrophenyl EGTA, with high intensity UV illumination (~320 nm, 10 pulses s⁻¹) in 2-μm diameter spots. In situ Cx43, NFκB and Akt staining was carried out after fixation and permeabilization of the alveolus. We quantified Cx43 mRNA by qPCR in AMs sorted from BAL and lung tissue samples (Influx Cell Sorter, BD Biosciences). BAL and cell culture supernatant cytokines were analyzed by ELISA. Western Blot analyses and Co-immunoprecipitations were performed as previously described^{29 (link)}. siRNA was complexed with freshly extruded liposomes and intranasally instilled.