A Real-Time, Bioluminescent Apoptosis Assay

This chapter describes a real-time, bioluminescent apoptosis assay technique, which circumvents the well-documented "timing condundrum" encountered when employing traditional apoptosis detection chemistries after exposures with inducers of unknown potential. The assay continuously reports the translocation of phosphatidylserine (PS) from the inner membrane leaflet of a cell to the exofacial surface during apoptosis. This homogenous, no-wash, plate-based assay is made possible by two different annexin V fusion proteins, which contain complementing NanoBiT™ luciferase enzyme subunits, a time-released luciferase substrate, and a fluorescent membrane integrity reagent. During apoptosis, luminescence signal is proportional to PS exposure and fluorescence intensity correlated with the degree of secondary necrosis. Altogether, the measures provide exquisite kinetic resolution of dose- and agent-dependent apoptotic responses, from early through late phases. At exposure termination, other compatible reagents can be applied to measure additional orthogonal correlates of cell health.