Gelatin Zymography for MMP-2/-9 Analysis

MMP-2/-9 activity was assayed using gelatin zymography as described previously3 (link). MSK QLL1 and SCC QLL1 cells were treated with gas (He+O₂) only, 1 kV of NTP for 1 s, 10 μg/ml of cetuximab, and NTP (1 kV) plus cetuximab (10 μg/ml), and incubated for an additional 24 h. The supernatant (100 μl) from each sample was mixed with 1 μl of 100 mM 4-aminophenylmercuric acetate, and the samples were activated for 1 h at 37°C. Next, each sample was placed in sample buffer for 10 min and electrophoresed in polyacrylamide gels at 125 V for 120 min at 4°C using a Novex Xcell II system (Life Technologies, Carlsbad, CA, USA). The gels were incubated in renaturation buffer for 60 min at room temperature, followed by incubation for 18 h in 100 ml of developing buffer at 37°C with light shaking. The gels were then stained for 3 h with Coomassie brilliant blue. After decolorization in 400 ml of methanol, 100 ml of acetic acid, and 500 ml of distilled water, images were obtained using an image analyzer.