Apopcyto caspase 3 colorimetric assay kit

The caspase-3 activity was quantified with an APOPCYTO Caspase-3 Colorimetric Assay Kit (MBL International Corp., Nagoya, Japan) according to the manufacturer’s instruction. In brief, the indicated cells were harvested and lysed. Cell lysate (20 μl) was added to the buffer containing Ac-DEVD-pNA to yield a 100 μl total reaction volume, and the mixture was incubated for 1 h at 37 °C. Optical density of each well was evaluated at 405 nm using a Bio-Tek microplate reader (Bio-Tek Instruments). The concentrations of released pNA were quantified based on the absorbance and calibration curve. The caspase-3 activity in the control group is deemed as 1.

Cell apoptosis was evaluated in terms of catalytic activity of Caspase-3 by using the APOPCYTO Caspase-3 Colorimetric Assay Kit (MBL International Corporation, Woburn, MA, USA), according to manufacturer’s protocol. Briefly, at 96 h after transfection, cells were detached, lysed and extracted proteins were incubated with the substrate N-acetylAsp-Glu-Val-Asp-AMC (DEVD-AMC). The hydrolysis of the proper substrate was evaluated through spectrofluorometry with 380-nm excitation and 460-nm emission filters by using POLARstar OPTIMA plate reader (BMG Labtech, Ortenberg, Germany). For terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, transfected cells were fixed and treated by using the In Situ Cell Death Detection Kit (Roche) according to manufacturer’s instructions. The cells were subjected to FACS analysis (BD Accuri™ C6 Cytometer, Becton Dickinson, Basel CH) and data were reported in graph as the percentage of positive cells.