2104 envision reader

Caspase-3/7 activity was assessed by the Caspase-Glo® 3/7 Assay (Promega). Cells (3000 per well) were seeded, allowed to adhere overnight and exposed to erlotinib (50nM) for 24h in the absence of TGF-β. Caspase-Glo® 3/7 was added to the cells and incubated for 30 minutes at room temperature per manufacturer's instructions. Caspase activity was measured by luminescence measurement using a 2104 EnVision reader (PerkinElmer).

We developed a high throughput assay to screen a 115 000-compound library at UC Berkeley Drug Discovery Center (DDC) at the Center of Emerging and Neglected Diseases. The assay was optimized for 384 well black plates (corning 3573) with the total reaction volume of 25 μL, with equal volumes of protein and substrate (12.5 μL) and 0.5 μL of DMSO or compound (final concentration of 40 μM) dissolved in DMSO which was pre-plated with an Analytik-Jena Cybio liquid handler which was also used to add protein and substrate reagents later during the actual run. Protein and substrate were diluted in same buffer used in 4.2 with exception of Antifoam (Spectrum chemicals, Cat# A1302) with the ratio of 1 : 5000 that was added to reduce surface tension. The fluorescent emission intensity was measured at the intervals of 0, 10, 20, 30 and 60 minutes using a 2104 Envision reader (PerkinElmer; excitation: 360 nm and emission: 460 nm). The 60 minute time point yielded the best Z prime and was chosen as the end point for all screening. The data was analyzed using dose response curve models (4 parameter fit).