Caspase 3 activity kit

Mangiferin-induced apoptosis of OVCAR3 cells was also assessed by measuring the activity of caspase-3. OVCAR3 cells were seeded into a 6-well culture plate at a density of 4×10⁵ cells/well for 16 h incubation. Subsequently, cells were treated with 25 µg/ml mangiferin for 0, 12, 24, 36, 48 h. Caspase-3 activity was measured by using a caspase-3 activity kit (Beyotime Institute of Biotechnology, Haimen, China). In brief, cells were lysed, the supernatant was collected, quantified, and incubated with the caspase-3-specific color substrate Ac-DEVD-pNA. Caspase-3 activity was determined by measuring optical density at OD400 nm.
Mitochondrial membrane potential was measured by fluorescent dye rhodamine-123. After treatment with 25 µg/ml mangiferin for 12, 24, 36 and 48 h, cells were collected and suspended in 1 ml of PBS containing 1 µg/ml rhodamine-123 and incubated at 37°C for 15 min. The fluorescence intensity of the cells was analyzed on a FACS Aria II cytometer (Becton Dickinson). All experiments were carried out in triplicate. Experiments were repeated three times, and data are representative of replicate experiments.

The activity of caspase-3 was determined using the Caspase-3 activity kit (Beyotime
Institute of Biotechnology, Haimen, China). To evaluate the activity of caspase-3, cell
lysates were prepared after their respective treatment with various designated treatments.
Assays were performed on 96-well microtitre plates by incubating 40 µl
protein of cell lysate per sample in 50 µl reaction buffer (1% NP-40,
20 mM Tris-HCl (pH 7.5), 137 mM Nad and 10% glycerol) containing 10 µl caspase-3 substrate
(Ac-DEVDpNA) (2 mM). Lysates were incubated at 37 °C for 2 h. Samples were measured with a
microplate reader (SPECTRA FLUOR, Austria) at an absorbance of 405 nm and it represented
the caspase-3 activity of this sample.

Caspase-3 activity was determined to investigate the apoptosis of cells. Following treatment, H9C2 cells cultured at a density of 1×10⁶ cells/well in 6-well plates were homogenized using 50 mM of potassium phosphate buffer. Following centrifugation at 10,000 × g at 4°C for 10 min, the supernatant was analyzed using the caspase-3 activity kit (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer's protocols. Briefly, 50 µl supernatant was mixed with 10 µl caspase-3 substrate Ac-DEVD-pNA and 40 µl buffer solution, and then incubated at 37°C for 2 h, and absorbance at 450 nm was measured, reflecting cleavage of the colorimetric substrate. Finally, values were normalized to those of the control group.