Apoptosis Marker Assays for HTS

A variety of assays are available to detect apoptosis; however, few are conveniently adapted for high-throughput screening (HTS) using a multimode plate reader. The markers most commonly used for in vitro detection of apoptosis include caspase-3/7 activity and phosphatidylserine (PS) exposure on the outer leaflet of the cell membrane. Caspase-3/7 activity is routinely measured using consensus tetrapeptide substrates in lytic cell-based assays to generate a fluorescent or luminescent signal measured with a plate reader. PS exposure historically has been measured using flow cytometry to detect binding of fluorescently-tagged annexin V; however, sample throughput and washing steps to remove unbound fluorescent probe have limited broad adoption of the flow cytometric approach for HTS. Recently, recombinant annexin V fusion proteins engineered to contain subunits of a shrimp-derived luciferase have been used to develop a no-wash enzyme complementation approach for detecting PS exposure using a multimode plate reader. This homogeneous annexin V-binding assay approach has broadened the availability of assays compatible with ultraHTS.