Category: Cellular Technologies

1362-C - Real-Time High Throughput Detection of Annexin V Binding and Caspase-3 Activity Using a Plate Reader

Tuesday, February 6, 2018
2:00 PM - 3:00 PM

Apoptosis (or programmed cell death) is a transient process that can occur quickly in some cells and more slowly in other members of the same population. Existing methods to measure apoptosis in a high throughput format are endpoint assays that kill the cells and provide a “snapshot in time” of what has happened in the cell population before the lytic detection reagent or fixative was added. The ability to repeatedly monitor the progress of apoptosis in a population of cells as it is occurring would provide useful additional information not available from endpoint assay methods that kill cells. To overcome these deficits and provide methods to improve the efficiency of assay development for HTS we developed two homogeneous real-time assays for detecting apoptosis that can be recorded using a plate-reading luminometer. The first assay is based on binding of annexin V to phosphatidyl serine (PS) which becomes exposed on the outer leaflet of the cell membrane during apoptosis. We engineered two fusion proteins composed of annexin V linked to a small or large subunit of an engineered shrimp luciferase. When the fusion proteins bind to PS in close proximity on the surface of apoptotic cells, the reconstituted luciferase generates light to report apoptosis in real-time. Multiplexing with a DNA binding dye (that only penetrates and stains dead cells with a compromised membrane) reports secondary necrosis in real-time from the same sample. The second real-time apoptosis assay approach relies on expression of a modified firefly luciferase biosensor containing the DEVD amino acid sequence that is cleaved by caspase-3. The biosensor luciferase exists in an inactive conformation in viable cells. Upon induction of apoptosis, caspase-3 cleavage of the DEVD sequence enables the luciferase to fold into an active conformation and generate a luminescent signal in real-time as the population of cells undergoes apoptosis. These real-time homogeneous apoptosis assays represent an improvement over endpoint methods by providing kinetic data from the same sample of live cells in real-time using a standard plate reading luminometer.

Terry Riss

Global Strategic Manager
Promega Corporation
Madison, Wisconsin

Dr. Terry Riss started the Cell Biology program at Promega in 1990 and held several R&D and Project Management positions since. Dr. Riss managed development of cell viability, cytotoxicity, apoptosis, and protease assay systems and also lead efforts to identify and promote multiplexing of cell-based assays to determine the mechanism of cell death. Dr. Riss now serves as Global Strategic Manager, Cell Health involved in outreach educational training activities. Dr. Riss has participated in several NIH study sections reviewing HTS grants and is co-editor of the In Vitro Cell Based Assays section of the Assay Guidance Manual hosted by NIH.