Methamphetamine induces autophagy as a pro-survival response against apoptotic endothelial cell death through the Kappa opioid receptor

Ma, J; Wan, J; Meng, J; Banerjee, S; Ramakrishnan, S; Roy, S

Caspase activation. Caspase-8, Caspase-3/7 and Caspase-9 activities were analyzed using Caspase-Glo luminescent-based assays according to the manufacturer's instructions (Promega Corporation, Madison, WI, USA). Cells (1 Â 10 4 ) were seeded into 96-well white opaque plates and a corresponding optically clear 96-well plate, then allowed to adhere overnight. The next day, MEK1 inhibitor PD98059 (20 mM) was added 2 h before the cells were treated with METH for 24 h. At the end of the incubation time, 100 ml of the appropriate Caspase-Glo reagent was added to each well containing 100 ml of blank, negative control or treated cells in culture medium. Plates were gently mixed and incubated for 1 h at room temperature. The luminescence was then read in a FLUOstar OPTIMA Microplate Reader (BMG LABTECH, Cary, NC, USA). Caspase activity was normalized to vehicle treatment.