Colorimetric protease assay

HL-60 cells (2×10⁶ cells/well) were incubated with A398 (6 µM) or etoposide (5 µM) for 1 h, 3 h, 6 h and 12 h. The activities of the caspases were carried out using colorimetric protease assay (Invitrogen, USA) following the manufacturer's protocol. Each kit contains a specific substrate: IETD (Ile-Glu-Thr-Asp), LEHD (Leu-Glu-His-Asp) and Ac-DEVD (acetyl-Asp-Glu-Val-Asp) for caspases -8, -9 and -3, respectively. Such substrates are labeled to the chromophore p-nitroanilide (pNA), which is released when they are cleaved by activated caspases and measured at 405 nm in a spectrophotometer (Biotek Instruments EL800, USA).

Molt-4 cells were incubated with D-carvone for 24 h. The activities of the caspases were carried out using colorimetric protease assay (Invitrogen, USA) following the manufacturer's protocol. Each kit contains a specific substrate: IETD (Ile-GluThr-Asp), LEHD
(Leu-Glu-His-Asp) and Ac-DEVD (acetyl-AspGlu-Val-Asp) for caspases -8, -9 and -3, respectively. Such substrates are labeled to the chromophore p-nitroanilide (pNA), which is released when they are cleaved by activated caspases and measured at 405 nm in a
spectrophotometer (Biotek Instruments EL800, USA).

The protease activity in plant extracts after blanching heat treatment and in untreated controls was determined using a colorimetric protease assay (Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to the manufacturer’s instructions. Samples were diluted in the range 1:5–1:40 to adjust the TSP concentrations to the same order of magnitude and were then measured in triplicate in 96-well plates. Six trypsin standards with concentrations of 0.005–500 mg L⁻¹ were used to build duplicate standard curves. For each sample and standard, 100 µl of succinylated casein solution was pipetted into one well and 100 µl of working buffer into another as a blank, before transferring 50 µl of the sample to both. The plates were incubated for 20 min at 22°C before adding 50 µl of working solution to each well and were then incubated for an additional 20 min at 22°C. The absorbance in each well was measured twice at 450 nm using an EnSpire multimode plate reader, and the data were exported in EnSpire Manager v4.13 (Perkin Elmer, Waltham, Massachusetts, USA).