Abstract
Hepatotoxicity, including drug-induced liver injury, is frequently accompanied by cell death. The latter is typically driven by apoptosis or necrosis, which substantially differ based upon biochemical and morphological criteria. This chapter describes two commonly used methods to probe apoptotic and necrotic activities in adherent monolayer cultures of primary hepatocytes. The apoptosis assay uses a prototypical substrate of caspase 3, the main executor of apoptotic cell death, which can be cleaved, yielding a product that can be measured fluorimetrically. The second assay relies on the disruption of the cell plasma membrane, which typically occurs in necrotic cell death and that results in the extracellular release of cytoplasmic enzymes, such as lactate dehydrogenase. The latter can be indirectly assessed by spectrophotometrically measuring the consumption of reduced nicotinamide adenine dinucleotide.
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Acknowledgements
This work was financially supported by the grants of the Agency for Innovation by Science and Technology in Flanders (IWT), the University, Hospital of the Vrije Universiteit Brussel—Belgium (Willy GeptsFonds UZ-VUB), the University of Sao Paulo—Brazil (USP), the Sao Paulo Research Foundation—Brazil (FAPESP), the Fund for Scientific Research—Flanders (FWO-Vlaanderen), the European Research Council (project CONNECT), the European Union (FP7) and Cosmetics Europe (projects DETECTIVE and HeMiBio).
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Maes, M. et al. (2015). Measurement of Apoptotic and Necrotic Cell Death in Primary Hepatocyte Cultures. In: Vinken, M., Rogiers, V. (eds) Protocols in In Vitro Hepatocyte Research. Methods in Molecular Biology, vol 1250. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2074-7_27
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DOI: https://doi.org/10.1007/978-1-4939-2074-7_27
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