Biochemical characterization of an in vitro model of hepatocellular apoptotic cell death

Mathieu Vinken, Elke Decrock, Elke De Vuyst, Luc Leybaert, Tamara Vanhaecke, Vera Rogiers

Research output: Contribution to journalArticlepeer-review

10 Citations (Scopus)


SCOPE: The present study was set up to critically evaluate a commonly used in vitro model
of hepatocellular apoptotic cell death. In this model, freshly isolated hepatocytes, cultivated in
a monolayer configuration, are exposed to a combination of Fas ligand and cycloheximide for
six hours. METHODS: A set of well-acknowledged cell death markers was addressed: (i) Cell
morphology was studied by light microscopy. (ii) Apoptotic and necrotic cell populations
were quantified by in situ staining with annexin-V, Hoechst 33342 and propidium iodide. (iii)
Apoptotic and necrotic activities were monitored by probing caspase 3-like activity and
measuring extracellular leakage of lactate dehydrogenase, respectively. (iv) Expression of
apoptosis regulators was investigated by immunoblotting. RESULTS: Initiation of apoptosis
was evidenced by activation of caspase 8 and caspase 9, and increased annexin-V reactivity.
Progression through the apoptotic process was confirmed by activation of caspase 3 and Bid,
enhanced expression of Bax, and the occurrence of nuclear fragmentation. Late transition to a
necrotic appearance was demonstrated by an increased number of propidium iodide-positive
cells and augmented extracellular release of lactate dehydrogenase. CONCLUSIONS: The in
vitro model allows to study the entire course of Fas-mediated hepatocellular apoptotic cell
death, which is not possible in vivo. This experimental system can serve a broad range of in
vitro pharmaco-toxicological purposes, thereby directly assisting in the reduction of animal
Original languageEnglish
Pages (from-to)209-218
Number of pages10
JournalATLA. Alternatives to Laboratory Animals
Publication statusPublished - 1 Apr 2009


  • primary hepatocyte
  • in vitro model
  • apoptosis


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