RESUMEN
High-throughput screening assays with multiple readouts enable one to monitor multiple assay parameters. By capturing as much information about the underlying biology as possible, the detection of true actives can be improved. This report describes an extension to standard luciferase reporter gene assays that enables multiple parameters to be monitored from each sample. The report describes multiplexing luciferase assays with an orthogonal readout monitoring cell viability using reduction of resazurin. In addition, this technical note shows that by using the luciferin substrate in live cells, an assay time course can be recorded. This enables the identification of nonactive or unspecific compounds that act by inhibiting luciferase, as well as compounds altering gene expression or cell growth.
Asunto(s)
Genes Reporteros , Ensayos Analíticos de Alto Rendimiento , Luciferasas/genética , Luciferasas/metabolismo , Antiinfecciosos/farmacología , Compuestos de Benzalconio/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Cicloheximida/farmacología , Luciferina de Luciérnaga/análisis , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Cinética , Oxazinas/metabolismo , Xantenos/metabolismoRESUMEN
A study comparing five different cAMP detection technologies in terms of sensitivity, robustness, and feasibility for HTS is presented. In this report, the following methods are described: a nonhomogeneous DELFIA, and the homogeneous methods based on time-resolved fluorescence (HTRF), luminescent singlet oxygen channeling or ALPHAScreen, FP, and high-affinity enzyme complementation. DELFIA had the highest sensitivity, whereas ALPHAScreen and HTRF shared several advantages, including high sensitivity, broad dynamic range, and minimal reagent addition steps. For G(s)-coupled antagonist screens, we found HTRF and ALPHAScreen the more sensitive and HTS-compatible techniques.