Caspase 3/7/10 Fluorometric Analysis

Briefly, 1 × 10⁶ HeLa cells were seeded in six-well plate. ASE was added with the increasing concentrations (25, 50, and 100 μg/ml), whereas untreated cells served as uninduced control. After 48 h of treatment, cells were centrifuged at 2,000 rpm, washed with PBS, and the cell pellet was stored at −20°C.

The Caspase 3, 7, and 10 Apoptosis fluorometric Assay Kit (G-Biosciences) was used to estimate the apoptotic activity of ASE on HeLa cells. Manufacturer’s instructions were followed to perform the assay. Briefly, the cell pellet was resuspended in 100 µL of lysis buffer and freeze/thawed for 4 to 5 times to obtain cell lysate. For each fluorometric reaction, 50 µL of cell lysate, 50 µL of 2X assay buffer containing 1 mM of DTT and 5 µL of 1 mM substrate “Aspartic acid–Glutamic acid–Valine–Aspartic acid–7-amino–4-trifluoromethyl coumarin” (DEVD-AFC) was added.

Caspases cleaved DEVD-AFC to release the peptide (DEVD) and fluorescent molecule (AFC), which can be read at emission 375 nm and excitation 530 nm. Untreated cells served as uninduced control containing 100 µL of assay buffer and 5 µL substrate AFC-DEVD. Two hundred µM of Z-VAD-FMK (Broad spectrum apoptosis inhibitor) added to 100 μg/ml ASE-treated cell lysate served as a negative control.

The reaction was carried out in 96-well plate. After taking readings at “t = 0,” reaction was incubated at 37°C for 60 min. The change in fluorescence absorbance at time point “0” minutes and time point “60” minutes was plotted in the graph.