Objective

We set out to develop a high throughput homogeneous method for detecting apoptosis in real-time by repeatedly recording data from the same sample of cells using a plate-reader. Previous methods to follow the progress of apoptosis required preparing several samples and performing endpoint assays. We modified the fluorescent annexin V phosphatidylserine (PS) binding assay by using a luciferase enzyme complementation approach that resulted in eliminating wash steps. We engineered two genetic fusion proteins composed of annexin v linked to small or large fragments of luciferase. The fusion proteins and a luciferase substrate are added as a reagent to the medium of cultured cells. When exposed to apoptotic cells, the annexin V-luciferase fragment fusion proteins bind to PS in close proximity to reconstitute an active luciferase and generate light. Cells plus reagent can be incubated up to 48 hours enabling real-time detection of the onset of apoptosis. The luminescent assay has been multiplexed with a fluorogenic DNA binding dye to simultaneously demonstrate the kinetics of secondary necrosis. Imaging the luminescent signal enables creation of time lapse movies showing individual cells among a population undergoing apoptosis. This new homogeneous apoptosis assay represents a simplification and improvement over flow cytometry and endpoint assay methods by providing kinetic data from the same sample of cells in real-time using a standard plate reading luminometer