Development of an exo-cell assay for quantification of γ-secretase activity in cells. (a) Titration of CHAPSO detergent in the exo-cell assay. CHAPSO detergent was titrated to determine the optimal amount required for stimulating γ-secretase activity. The titration was performed using 10,000 HeLa cells and 1 μM Sb4 substrate. This reaction was incubated for 2.5 hours at 37°C. Supernatent was then collected and analyzed using ruthenylated G2-10* antibody. Activity was quantitated by measuring ECL. For each assay point n = 4, and s.d. is plotted. (b) Titration of the number of HeLa adenocarcinoma cells from which the exo-cell assay can detect γ-secretase activity. The indicated number of HeLa cells were seeded in a 96-well plate and allowed to attach overnight. The next day media was removed and replaced with fresh media containing 0.25% CHAPSO detergent, 1 μM Sb4 substrate, and DMSO or 1 μM Compound E to define background. Values plotted represent the activity quantified for each cell number assay point with GSI-defined background subtracted. For each assay point n = 4, and s.d. is plotted. (c) Dose-dependent inhibition of γ-secretase activity by GSI-34. HeLa cells were seeded 10,000 cells per well of 96-well plate. The cells were treated for 24 hours with the indicated concentration of GSI-34 inhibitor. Cells were then washed once with PBS and then the exo-cell assay was performed using 1 μM Sb4 substrate and 0.25% CHAPSO detergent. (d) IC50 values of distinct GSIs in extended exo-cell assay. IC50 values were obtained for 2 distinct GSI compounds using the extended exo-cell assay. For each data point n = 3, and s.d. is plotted.