Caspase colorimetric protease assay sampler kit

The Caspase Colorimetric Protease Assay Sampler Kit (Thermo Fisher Scientific) was used for the determination of caspase-3 and caspase-9's activities in HepG2 cells according to the protocol of the manufacturer [21 (link)]. In short, we cultured the HepG2 cells by utilizing 96-well plates at 1 × 10⁴ cells/well density, and then, these cells were put in 5 percent CO₂ wet air overnight. Complex 1 was then added for treatment as previously described for 24 h. Then, we gathered the treated and control cells, and these cells were then resuspended in cold cell lysis buffer (50 μL) on ice for 10 minutes. Through centrifuging for three minutes at 10,000 × g, we extracted the cytosolic fraction. A total of 50 μg cytosolic extracts were added into 96-well plates, and then, we added the reaction buffer of 50 μL which involves 10 mM dithiothreitol into these plates. Adding the caspase substrate of 5 μL aliquot into those and then culturing them for two hours at 37°C. On the microplate reader, the plate was read at 405 nm. In the research, the standard curve was drawn with PNA. All of the tests were repeated 3 times.

The Cell Death Detection ELISA^PLUS (Roche) was performed as described54 (link) using cells incubated for 24 or 48 h with purified OMVs LB226692 (4.7 μg/ml of protein containing 580 ng/ml of Stx2a) or C227-11Φcu (4.7 μg/ml of protein) or purified Stx2a (580 ng/ml) or OMV buffer. To identify the minimum apoptotic dose of OMV-associated and free Stx2a, cells were incubated (48 h) with LB226692 OMVs or purified Stx2a containing 580 ng/ml to 3.625 ng/ml of Stx2a. Apoptosis was quantified by flow cytometric detection of hypodiploid nuclei as described55 (link)56 (link). Staurosporin (1 μM) (Sigma) was a positive, and untreated cells a negative control.
Activities of caspase-2, -3, -6, -8, and -9 in lysates of cells treated (48 h) with purified OMVs LB226692 (4.7 μg/ml of protein containing 580 ng/ml of Stx2a) or the corresponding amount of OMVs C227-11Φcu protein were assayed with the Caspase Colorimetric Protease Assay Sampler Kit (Invitrogen). The caspase activities in OMV-treated cells were expressed as a fold-increase of their activities in untreated cells. If required, cells were pretreated (30 min) with 50 μM pan-caspase inhibitor z-VAD-fmk or caspase-9 (z-LEHD-fmk) or caspase-3 (z-DEVD-fmk) inhibitor (R & D Systems).