MMP-2 and MMP-9 gelatinase activity with zymography

NRK-52E cells were cultured with 10% FBS in a 6-well plate and seeded at 5 × 10⁵ cells/ml. 100 μg/mL of AGEs were treated to the kidney cell for 24 h, and then conditioned media were collected in a 1.5 mL microcentrifuge tube. After centrifuged (1300 rpm, 20 min, 4 °C), the supernatant was collected. The samples were electrophoresed on an 7.5% acrylamide gel containing 4 mg/mL gelatin from bovine skin (Sigma-Aldrich, St. Louis, Missouri, USA). After electrophoresis, the gel was washed with renaturing buffer for 30 min (2.5% Triton X-100, 50 mM Tris–HCl, pH 7.5), incubated with developing buffer (Tris Base 12.1 g, Tris HCl 63.0 g, NaCl 117 g, CaCl₂ 27.4 g added distilled water up to 1 L) for overnight at 37 °C, and washed with distilled water, and then stained with Coomassie blue. The presence of MMP-2 and MMP-9 gelatinolytic activity were detected on a blue background after destaining.