Figure 2

Application of perfluorodecalin for PCLS culture. (A-C): Comparison between mouse and rat PCLS: PCLS were generated from rat or mouse using a Krumdieck tissue slicer as described in Figure 1 and incubated for 0 and 72 h. Three independent experiments (6 slices for each sample) were performed. Slices were incubated either in Williams’ medium E (WME) or DMEM containing sodium pyruvate, glucagon, insulin, corticosterone, EGF and 5% dialyzed FCS. (A): Paraffin sections (5 μm thick) were prepared and stained with HE. An example of representative data 0 and 72 h after slicing is shown (bar represents 50 μm) and numbers of nuclei in hepatocytes per ten HPF from 4 independent animals (average of number of nuclei before ex vivo incubation as 100%) are shown. (B): ATP level was measured using ATP determination kit (Biaffin GmbH). (C): Triglyceride content in rat and mouse PCLS incubated in the WME medium for 72 h was measured using Triglyceride colorimetric assay kit and fold-increase from value before ex vivo incubation are shown. (D): PFD treatment delayed the loss of ATP levels in the mouse PCLS: PCLS were incubated 0, 24, 48 and 72 h. ATP levels were measured from each slice (12 slices from 4 independent experiments in each condition) and the percentage of ATP is represented (value before ex vivo incubation as 100%). (E): HE staining of paraffin sections (5 μm thick) obtained from 0, 48 and 72 h samples with (+) or without (−) PFD. Bar represents 50 μm. Numbers of nuclei in hepatocytes per ten HPF from 4 independent animals (average value before ex vivo incubation as 100%). P value: student’st-test.