Gelatin zymography assays of MMP-9 activity

BXPC-3 cells (1×10⁶ cells/well) were seeded in 6-well plates and cultured with DMEM in a humidified chamber at 37°C in 5% CO₂. BXPC-3 cells were treated with 0, 6.25, 12.5 and 25 µM paeoniflorin for 48 h, and the MMP-9 activity of BXPC-3 cells was determined by gelatin zymography assays. Equal volumes of sample (40 µl) were mixed with sodium dodecyl sulfate (SDS) sample buffer (Tiandz Inc., Beijing, China). The miscible liquids were subjected to 10% SDS-PAGE gels polymerized with 1 mg/ml gelatin (Tiandz Inc.). The gel was then washed three times for 20 min at room temperature in 2.5% Triton X-100 to remove SDS following electrophoresis. Next, the gel was incubated in radioimmunoprecipitation buffer (Nanjing KeyGen Biotech Co., Ltd.) at 37°C for 12 h. The gel was stained with 0.05% Coomassie brilliant blue stain R-250 (Beyotime Institute of Biotechnology) was used to stain followed by destaining with 7% acetic acid (Sinopharm Chemical Reagent Co., Ltd.).