Gelatin zymography

Conditioned medium from cultured cells was collected as previously described [22]. Briefly, cells were cultured in medium without serum for 24–48 hours at similar confluency. Upon collection, the culture medium was clarified by centrifugation at 6000rpm for 2 min. Then the conditioned medium was mixed with zymogram sample buffer (BioRad, Hercules, CA) and incubated at room temperature for 10 minutes. The conditioned medium was resolved on 7.5% SDS-PAGE gels containing 1 mg/ml gelatin. After electrophoresis, the SDS-PAGE gel was then transferred into 2.5% Triton X-100 and incubated at room temperature for 40 minutes with shaking. After incubation, the gel was rinsed with incubation buffer (50 mM Tris-B pH8.0; 150 mM NaCl; 10 mM CaCl; 0.05% NaN₃) before being soaked in incubation buffer at 37°C for 20~24 hours. After incubation, the gel was rinsed with dH₂O 3 times before staining with Coomassie blue for 40 minutes at room temperature. Finally, destaining was performed at room temperature for 2 hours before the gels were imaged. Active and Pro-MMP2 bands were quantified by the area density measurement using Chemi Doc-IT² Imager software (UVP).