Measurement of apoptotic and necrotic cell death in primary hepatocyte cultures

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.