Gelatin Zymography for MMP-2 and -9 Assessment

The activity of MMP-2 and -9 was assessed by gelatin zymography as previously described [8 (link), 27 (link)]. Protein concentration was determined by Pierce™ BCA assay kit (Thermo Scientific, Rockford, IL). Murine and human BALF and lung homogenates were separated by electrophoresis using 10% SDS-polyacrylamide gels containing 0.1% gelatin. Gels were then washed in 2.5% triton 100 renaturing buffer followed by overnight incubation in developing buffer. To visualize bands gels were stained with 0.5% Coomassie blue for 1 h and then destained with 40% methanol/10% acetic acid until clear bands were visualized. Densitometry was performed as previously described and the MMPs activity were normalized for total BALF and lung homogenates protein concentration.