Caspase3 activity assay kit

The Caspase-3 Activity Assay Kit (Elabscience, China) was utilized to carry out cell apoptosis detection. Then, 5×10⁴ cells/well were seeded in a 96-well plate. After that, 30 µL (each well) cell lysis buffer was added to the samples. The cells were subsequently placed on ice for 5 min. The next procedure involved the addition of 5 μL Ac-DEVD-pNA to the samples, followed by 4-h incubation. The activation of Caspase-3 was eventually determined using a spectrophotometer at 400 nm.

The Caspase-3 activity in
breast cancer cells, MDA-MB-231 and MCF-7, was examined using the
Caspase 3 Activity Assay Kit (Elabscience, E-CK-A311A) following the
manufacturer’s protocol. Briefly, MDA-MB-231 and MCF-7 cells
were seeded in a six-well plate at a density of 2 × 10⁵ cells and incubated overnight for 70–80% confluency. Post-incubation,
the cells were treated with respective IC₅₀ value of the
EA extract of C. gloeosporioides for
24 h. Post-treatment, the cells was trypsinized and washed once with
cold 1X PBS to remove excess culture media and trypsin. Further, the
cells were lysed with lysis buffer (containing DTT) for 30 min at
4 °C and centrifuged at 12,000 rpm for 15 min at 4 °C. The
supernatant containing Caspase-3 protein was collected, and the reaction
mixture was prepared by adding the protein sample, reaction buffer,
and substrate Ac-DEVD-pNA at a final volume of 100 μL and incubated
at 37 °C for 4 h. Post-incubation, the absorbance was recorded
at 405 nm using a Multiskan microplate spectrophotometer (Thermo Fisher
Scientific, USA), and the fold change in the activity of Caspase-3
was calculated. The representative result was from three independent
experiments.