Pentobarbital (100 mg/kg IP) was used for anesthesia prior to sacrifice. This was followed by the placement of a tracheotomy tube, and the lungs were first lavaged with 5 ml of sterile PBS, and the returned fluid was used for Cys/Cyss determination. For the isolation of alveolar macrophages, lungs were then lavaged with 10 ml of sterile PBS four times. The recovered sample was centrifuged at 1500 rpm (405 xg) for 7 minutes and the cell pellet was re-suspended for cell count determination using a hemocytometer. Following cell count determination, a smear was made to evaluate for cell types, which showed >90% macrophages. The macrophages were used for various experiments including electromobility shift assay, overnight in vitro studies, as well as for RNA extraction followed by RT-PCR and quantitative PCR to determine gene expression.
