4.8. Caspase-3, Caspase-8 and Caspase-9 Activities

After treating cells with UF for 24 h, the cells were harvested using cell scrapers and washed with ice-cold PBS. Then, the cells were lysed for 30 min on ice in 100 µL of cell lysis reagent supplemented with a complete protease inhibitor cocktail. The protein concentration of cell lysates was determined by a Bicinchoninic acid (BCA) assay. The Caspase-3,-8,-9 were tested using a Caspase-3, -8, -9 Activity Assay Kit, respectively, produced by the Beyotime company. The principle of the Caspase -3, -8, -9 activity test is that Caspase-3, -8, -9 could catalyze Ac-DEVD-pNA (acetyl-Asp-Glu-Val-Asp p-nitroanilide), Ac-IETD-pNA(acetyl-Ile-Glu-Thr-Asp p-nitroanilide), and Ac-LEHD-pNA (acetyl-Leu-Glu-His-Asp p-nitroanilide) to produce pNA (p-nitroaniline), respectively. pNA exhibits a strong abosorption at 405 nm.