Multi well reader

Caspase activity was measured as described previously (Kim et al., 2015 (link)). The cells were harvested, washed with cold PBS, and lysed in lysis buffer (R&D Systems, Inc., Minneapolis, MN, USA) on ice for 20 min. The cell lysate was centrifuged at 10,000×g for 2 min and 100 µg of total protein was quantified. The quantified protein was mixed with 2X reaction buffer and colorimetric tetrapeptides, Z-DEVD, Z-IETD, and Ac-LEHD, for caspase-3, -8, and -9, respectively. After the reaction mixture was incubated at 37°C for 2 h, the enzymatic release of p-nitroaniline (pNA) was confirmed by measurement at 405 nm using a multi-well reader (Thermo Fisher Scientific).

The activities of caspases in CRC cells were detected as previously described (Choi et al., 2015 (link)). The cells were incubated with the lysis buffer (R&D Systems, Minneapolis, MN, USA) for 10 min on ice. Total protein (100 μg) samples were incubated with 2x reaction buffer and substrates of colorimetric tetrapeptides, Z-DEVD, Z-IETD, and Ac-LEHD for caspase-3, -8, and -9, respectively. The reaction mixtures were incubated at 37°C for 2 h, and then the enzyme-catalyzed release of p-nitroaniline was quantified at 405 nm using a multi-well reader (Thermo Fisher Scientific).

Cell viability was measured by evaluating the metabolic activity of mitochondria in living cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Amresco), as previously described (Kim et al., 2015 (link)). The MTT assay was conducted using mitochondrial enzymes to reduce yellow tetrazolium MTT to purple MTT formazan. Cells were seeded on culture plates and treated with or without diverse reagents in each growth medium for the indicated concentrations for 24 or 48 h. The reagent-treated cells were further cultured for 2 h by adding MTT (0.5 mg/mL) in the dark. The formazan granules produced by living cells were dissolved in DMSO, and the absorbance was measured at 540 nm using a multi-well reader (Thermo Fisher Scientific, Vantaa, Finland). The percentage of viable cells was calculated according to the formula [(treated group/control group)×100].