Caspase-3 and −9 colorimetric protease assay

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 caspase-3 and −9 activity was determined using a colorimetric protease assay. In accordance with the manufacturer's protocol (Nanjing KeyGen Biotech Co., Ltd.), BXPC-3 cells lysates were prepared in cell lysis buffer for 30 min on ice and centrifuged at 12,000 × g for 15 min at 4°C. Supernate was collected and the protein concentrations were determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Equal amounts of protein (40 ng) were mixed with reaction buffer (Ac-DEVD-pNA for caspase-3, Ac-LEHD-pNA for caspase-9) and incubated at 37°C for 2 h in the dark. Caspase-3 and −9 activity was then measured using a microplate reader (ELX800; Bio-Tek) at an absorbance of 405 nm.