Apotarget caspase 3 colorimetric protease assay kit

Proteolytic activity of caspase-3 in lysates of monocytes was determined using the ApoTarget™ Caspase-3 Colorimetric Protease Assay kit (Invitrogen). Human monocytes (5 × 10⁶) were lysed and cytosolic extract (200 μg) was incubated at 37 °C for 2 h with 200 μM substrate, DEVD-pNA, is composed of the chromophore, p-nitroanilide (pNA), and a synthetic tetrapeptide, DEVD (Asp-Glue-Val-Asp), which is the upstream amino acid sequence of the caspase-3 cleavage site in poly (ADP-ribose) polymerase. Upon cleavage of the substrate by caspase-3-free pNA light absorbance was quantified using a microplate reader at 405 nm.

The caspase 3 activity was assayed using the ApoTarget Caspase-3 Colorimetric Protease Assay Kit (KHZ0022; Invitrogen) according to the manufacturer's protocol. Briefly, 5×10⁶ thyroid cancer cells were treated with lidocaine and bupivacaine with or without specific inhibitors for the indicated time periods. The cells were harvested using trypsinization and cell lysates prepared as described above. Samples of the cell lysates (100 μg protein per sample) were mixed with reaction buffer and 200 μM substrate (DEVD-pNA) and incubated for 2 hours at 37°C in the dark. The absorbance was then measured at 405 nm and the sample readings calculated by subtracting the absorbance of blank samples.