Secreted protein samples from purified neutrophils (2 × 107 cells/ml) were examined by ELISA, activity assays and immunoblots. NE levels were determined by ELISA (R&D Systems). Cathepsin G activity was determined in samples with a colorimetric cathepsin G activity assay kit (Abcam, ab126780, Cambridge, MA), as described by manufacturers. Immunoblots were performed with anti-human lactoferrin polyclonal rabbit (immunogen was full length native protein, Cat. # ab15811, Abcam) and anti-human hCAP18 rabbit polyclonal antibodies (Cat. # PA5-20513, Invitrogen). BALF gelatinase activity (MMP9) was determined by gelatin zymography66 (link). Densitometry was performed on MMP9 positive bands and represented densitometry units (DU) measured by pixel intensity of bands, using Bio-Rad Laboratories Image Lab software (version 4.0, build 16). Equal cell numbers in each experiment was demonstrated by profiling electrophoretic of whole cell lysates in a Coomassie blue stained gel, i.e. loading control. O2− production was quantified by O2− dismutase–inhibitable reduction of cytochrome c at 550 nm, as previously described67 (link).
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