*4.11. Caspase-3 Activation Assay*

The measure of caspase-3 activity was performed using a commercially available kit, according to the manufacturer's instructions (BD Pharmingen). At 50% confluence, PC12 cells were cultured in the presence of DEHP alone (85 μM), HE alone (1 μM), or DEHP with HE at 37 ◦C for 24 h. Cells were harvested and incubated in lysing buffer for 30 min and then incubated with 20 μM Ac-DEVD-AMC (a substrate for caspase-3-like proteases) at 37 ◦C for 1 h. The release of aminomethylcoumarin (AMC) was then measured on a Perkin-Elmer fluorimeter using an excitation/emission wave length of 380 nm/460 nm. The results were corrected for total protein content in the lysates using the bicinchoninic acid assay (Pierce, Rockford, IL, USA), and expressed as the percentage of activity in lysates from control cultures.
