Caspase 3 colorimetric protease assay kit

For measurement of caspase activity, 1 × 10⁴ cells/well were seeded and exposed to different combination of treatments for 24h. For each treatment condition, cells were also exposed to caspase inhibitor Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]—fluoromethylketone, 50μM, Promega), a cell-permeable pan caspase inhibitor that irreversibly binds to the catalytic site of caspase proteases and inhibit induction of apoptosis. The caspase inhibitor was added 1h prior to treatments. The activity of caspase-3 was then measured using caspase-3 colorimetric protease assay kit (Invitrogen) following the manufacturer’s protocol. Briefly, the cell lysate was collected in RIPA buffer and the concentration of protein was determined using Bradford assay. Equal amount (60 μg) of protein mixed with reaction buffer was added to microtiter plates following incubation with caspase-3 substrate (acetyl-Asp-Glu-Val-Asp p-nitroanilide, Ac-DEVD-pNA) for 1 h. The absorbance was read at 405 nm using an micro-plate reader (Start-fax 2100, Awareness Tech. Ltd). The colorimetric assay is based on the hydrolysis of caspase-3 substrate by caspase-3 enzyme, resulting in the release of the p-nitroaniline (pNA) moiety. The concentration of the pNA released from the substrate was calculated from the absorbance values at 405 nm.

Approximately 2.5 × 10⁵ cells were seeded in a 6-well plate for estimation of caspase activity through caspase-3 colorimetric protease assay kit (Invitrogen). Briefly, proteins were extracted using RIPA buffer; Bradford assay was used to measure protein concentration and then the equal amount (60 μg) of protein was added to microtiter plates with a caspase-3 substrate (Ac-DEVD-pNA) and thereafter absorbance was measured at 405 nm in Multiscan plate reader.

Caspase-3 activity of cells was determined using a Caspase-3 Colorimetric Protease Assay Kit (#KHZ0021, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Briefly, after 72 hours of treatment, cells were re-suspended in chilled cell lysis buffer and incubated on ice for 10 min. After 1 min centrifugation at 10000 x g, protein concentrations of the supernatants (cytosol extract) were measured using BCA Protein Assay Kit (#B6916, Sigma, St. Louis, MO, USA). Equal amounts of proteins were then incubated at 37°C with Reaction Buffer containing 10 mM DTT and 200 uM DEVD-pNA substrate for 2 hours. The optical density was measured at 405 nm with a Thermo Scientific Varioskan Flash plate reader (Thermo Fisher Scientific, Waltham, MA, USA).

After OGD, the PC12 cells were lysed with lysis buffer (Thermo Scientific, Waltham, MA, USA). Following two freeze–thaw cycles, the cell lysates were centrifuged at 21,500×g (Heraeus Multifuge 1S-R with Microliter Rotor, Thermo Scientific) for 3 min at 4 °C. The supernatants were subsequently collected and stored at –80 °C prior to being analyzed. The caspase-3 activity of the lysates was measured using a caspase-3 colorimetric protease assay kit (Thermo Scientific). The results were expressed as the relative percentage of caspase-3 activity of each sample compared with the control cells.

Caspase activity was assessed using Caspase 8 Colorimetric Protease Assay Kit or Caspase 3 Colorimetric Protease Assay Kit, respectively (both purchased by Thermo Fisher Scientific, Carlsbald, CA, USA), according to manufacturer’s instructions. Briefly, after 24 h treatment of 3 × 10⁶ cells/sample with HIHE soxhlet, cells were washed in PBS 1X and suspended in Cell Lysis Buffer on ice for 10 min. Cellular lysates were centrifuged, collected, and normalized in terms of protein concentration, according to Bradford assay [45 (link)]. Cellular lysates were then incubated for 2 h at 37 °C in the dark with 2X Reaction Buffer, containing DTT 10 mM and 200 µM of caspase 8 or caspase 3 substrate. Both substrates consist of a synthetic tetrapeptide, IETD (Ile-Glu-Thr-Asp) specific for caspase 8, and DEVD (Asp-Glu-Val-Asp) specific for caspase 3, conjugated with the chromophore p-nitroanilide (pNA). In presence of caspases’ activity, the specific substrate is cleaved from the chromophore and free pNA is used as a reporter, whose absorbance is measured at 405 nm, using the microplate reader Victor X3 (Perkin Elmer). Caspase activity was expressed as the fold increase of treated cells compared to untreated cells.