*4.5. Zymography*

MMP-2 and MMP-9 activities in the conditioned medium of IL-1β-treaded cells (10 ng/mL for 24 h) were analyzed by zymography. The medium was lyophilized and concentrated three times. Novex® 10% Zymogram (Gelatin) Protein Gels, 1.0 mm, 15-wells (Life Technologies) were used for electrophoresis. After 1.5 h of electrophoresis, the gel was incubated for 30 min at room temperature in renaturing buffer (Life Technologies) with gentle agitation. The buffer was replaced with developing buffer (Life Technologies) for 30 min at room temperature and subsequently replaced for fresh buffer. After overnight incubation, the gels were washed with demi water and stained for 1 h with SimplyBlue SafeStain (Life Technologies). MMP-2 and MMP-9 activity was displayed as unstained bands.
