Caspase 8 colorimetric assay kit

The activities of caspase 3 and 8 were measured using APOPCYTO Caspase-3 Colorimetric Assay Kit (Medical and Biological Laboratories) and Caspase-8 Colorimetric Assay Kit (BioVision), respectively. Briefly, total cell protein was extracted using ice-cold cell lysis buffer. Then, 100–200 μg of total protein was diluted in 50 μl of lysis buffer and 50 μl of 2× reaction buffer containing 10 mM DTT. 5 μl of caspase 3 or 8 substrate were added into each well of a 96-well microplate. After incubation at 37°C for 3 hours, the absorbance was measured at 405 nm.

The procedure was conducted according to the instructions provided by the kit manufacturer (Caspase-8 Colorimetric Assay Kit, BioVision K113; Milpitas, CA, USA). A2780 and A2780cis cells were seeded in 6-well test plates (3 × 10⁵ cells per 1 mL of medium and 3 mL of suspension per well). The procedure was conducted after 24 h. The tested compounds were added to the cell cultures (one sample in each experiment was not treated with the compounds, for control) and incubated for 2 and 4 h. Then the cells were collected by means of trypsinization and centrifuged (10 min, 1500 rpm, 4 °C), rinsed with PBS + 0.01 M EDTA, and centrifuged again. The cell pellets were suspended in 0.055 mL of cell lysis buffer, stirred vigorously, incubated on ice for 10–20 min, and centrifuged (10 min, 12,000 rpm, 4 °C). In case of each assay, 0.050 mL of the supernatant (cell lysate) was collected and mixed with 0.050 mL of reaction buffer supplemented with dithiothreitol and IEDT-pNA (a substrate for caspase-8). The samples were then incubated at 37 °C, protected from light. Then the samples were diluted to the volume of 0.9 mL with a dilution buffer and absorbance was measured (λ = 405 nm). The results are showed as the mean of at least three independent experiments ± SD.

Cells were seeded on 6-well plates. After indicated stimulation, cells were lysed in 50 µL of Cell Lysis Buffer included in the Caspase-8 Colorimetric Assay Kit (Biovision, Milpitas, CA, USA). Ten microliters of soluble cell extracts was mixed with 100 µL of caspase reaction buffer (10 mM Tris-HCl pH7.4, 150 mM NaCl, 0.1% CHAPS, 2 mM MgCl2, 5 mM EGTA, and 1mM DTT) supplemented with the caspase-8-specific substrate IETD-pNA (Biovision) at a final concentration of 100 µM and incubate at 37 ℃ for 1h. Cleaved pNA was measured using a microplate reader with the absorbance was read at 405 nm. Data are normalized to control (100%) without stimulus.