2.3. Hepatocyte Isolation Procedures and Incubations

Hepatocytes were prepared from anesthetized mice (isoflurane inhalation) using the collagenase perfusion method of Geohagen et al. (2016). Briefly, to separate dead hepatocytes, isolated cells were centrifuged (140g × 8 mins) in a Percoll gradient and then washed in media (140g × 3 mins) to remove Percoll. The isolation procedure yielded ~30–40 million cells with 80–90% viability as determined by trypan blue exclusion. Hepatocytes (100,000 cells/ml) were incubated in covered 35mm plastic dishes containing supplemented RPMI-1640 media at 37°C in a humidified atmosphere of 95% O₂/5% CO₂. The concentration-dependent cytoprotection (0.01–3.0mM) of individual test compounds was determined in isolated hepatocytes exposed to APAP (e.g., 1 mM × 4 hrs incubation). Control conditions included: vehicle alone (0.1% DMSO in media), vehicle plus APAP (e.g., 1 mM × 4 hrs incubation) and cytoprotectants alone (3.0 mM × 4 hrs). As sensitive indices of hepatocyte injury (Geohagen et al., 2016; Zhang et al., 2013), we measured the respective activities of lactate dehydrogenase (LDH) and the liver specific enzyme, alanine aminotransferase (ALT) in hepatocyte medium. The concentration-response data were fitted by nonlinear regression analyses (Geohagen et al., 2016). In all studies, hepatocyte viability and other toxic measures (see ahead) were determined in at least n = 4–6 independent experiments.