Abstract
This chapter describes a simple, nondestructive, annexin V apoptosis detection method that can be employed in real time over a 48-h test exposure. The real-time functionality allows for temporal resolution of apoptotic and cell death responses during the test exposure and obviates the need for onerous sample preparation and time course protocols associated with other annexin V methods. Further, this technique is eminently accessible to a wide range of laboratories because it does not require flow cytometry or other cytometric methods. It was developed for use with a variety of microplate well densities and with standard multimodal plate readers. The central feature of this assay is that it continuously reports the residency status of phosphatidylserine (PS) on the exofacial surface of a cell as it is translocated from the inner membrane leaflet during the apoptotic process. This homogenous, no-wash assay is made possible by two optimized and distinct annexin V fusion proteins which contain complementing NanoBiT™ luciferase enzyme subunits, a time-released luciferase substrate, and a fluorescent membrane integrity reagent. During an apoptotic event, the luminescent signal arising from an assay well is proportional to the number of cells with PS exposure, and fluorescence intensity correlates with the degree of cell death (secondary necrosis). Conversely, untreated cells contribute negligible luminescent or fluorescent signals throughout the time course. The data collected from these assay measures provide for both standard potency determinations and kinetic characterization of dose- and agent-dependent apoptotic responses, from early through late phases.
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Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35(4):495–516
Riss T, Moravec R (2004) Use of multiple assay endpoints to investigate the effects of incubation time, dose of toxin, and plating density in cell-based assays. Assay Drug Dev Technol 2:51–62
Niles A, Moravec R, Riss T (2009) In vitro viability and cytotoxicity testing and same-well multiparametric combinations for high throughput screening. Curr Chem Genom 3:31–41
Jessel R, Haertel S, Socaciu C, Tykhonova S, Diehl HA (2002) Kinetics of apoptotic markers in exogenously induced apoptosis of EL4 cells. J Cell Mol 6(1):82–92
Niles A, Moravec R, Riss T (2008) Update on in vitro cytotoxicity assays for drug development. Exp Op Drug Disc 3:655–669
Niles A, Riss T (2014) Multiplexed viability, cytotoxicity, and caspase activity assays. Methods Mol Biol Apoptosis Cancer:21–33
Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, Henson PM (1992) Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol 148:2207
Dive C, Gregory CD, Phipps DJ, Evans DL, Milner AE, Wyllie AH (1992) Analysis and discrimination of necrosis and apoptosis (programmed cell death) by multiparameter flow cytometry. Biochim Biophys Acta 1133:275
Kerr J, Wyllie A, Currie A (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. B J Cancer 26(4):239–257
Klion F, Schaffner F (1966) The ultrastructure of acidophilic “Councilman-like” bodies in the liver. Am J Path 48:755
Farbman AI (1968) Electron microscope study of palate fusion in mouse embryos. Devl Biol 18:93
van Meer G, Voelker DR, Feigenson GW (2008) Membrane lipids: where they are and how they behave. Nat Rev Mol Cell Biol 9:112–124
van der Mark VA, Elferink R, Paulusma CC (2013) P4 ATPases: Flippases in health and disease. Int J Mol Sci 14:7897–7922
Segawa K, Nagata S (2015) An apoptotic ‘eat me’ signal: phosphatidylserine exposure. Trends Cell Biol 25(11):639–650
Koopman G, Reutelinsperger C, Kuijten G, Keehnen R, Pals S, van Oers MH (1994) Annexin V for flow cytometric detection of phosphatidyl serine expression on B cells undergoing apoptosis. Blood 84:1415–1420
Kupcho K, Schultz J, Hurst R et al (2019) A real-time, bioluminescent annexin V assay for the assessment of apoptosis. Apoptosis 24(1–2):184–197
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Niles, A.L., Kupcho, K.R. (2022). A Nondestructive, Real-Time Annexin V Apoptosis Assay. In: Barcenilla, H., Diaz, D. (eds) Apoptosis and Cancer. Methods in Molecular Biology, vol 2543. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2553-8_1
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DOI: https://doi.org/10.1007/978-1-0716-2553-8_1
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