The software ignored partial nuclei at the borders of each image. Cy3 staining was used to identify further the nuclei of cell lineages of interest. Dabrafenib Using the ScanR analysis software, a mask was incorporated that defined Cy3-positive cells as those where cytoplasmic staining overlapped −5 pixels inside the DAPI-stained nucleus and extended +15 pixels outside the

nucleus. Data were displayed in histograms, so that cells were gated on the basis of intensity of Cy3 staining, preventing incorrect inclusion of Cy3-negative cells adjacent to Cy3- positive cells. This gate was maintained for each cell lineage. To identify telomeres in Cy3-positive cell nuclei, Cy5 Z maximum fluorescence intensity

was used with application of threshold object recognition and a watershed algorithm to outline individual telomeres, enabling maximum separation. These were depicted in histograms. Data obtained Gamma-secretase inhibitor for Cy3-positive nuclei and the subobject of individual telomeres in each nucleus were exported into Microsoft Excel spreadsheets. Telomere area was measured as the mean telomere area in pixels2 per nucleus. Nuclear area and nuclear density for each cell line were measured by the mean DAPI staining pixels2 for each nucleus and mean DAPI Z maximum fluorescence intensity, respectively. Statistical analysis was performed on GRAPH PAD Prism5 software (Graph Pad, San Diego, CA) and SPSS using a linear regression test as data demonstrated a normal distribution (Supporting Fig. 2). A P value of ≤0.05 was considered significant. Analysis of variance and Dunn’s multiple comparison tests were used to compare cell lineages. Five hyperoxalosis liver explants and sixteen age-matched time-zero livers were compared to determine whether organ acquisition, storage, or cold/warm ischemia influenced telomere length. Explanted hyperoxalosis livers were chosen because they had normal histology and PRKD3 were processed immediately with the shortest possible

ischemia time. There was no significant difference in telomere length or telomere number measured by Q-FISH for any cell lineage (Fig. 2). There was no evidence that cold or warm ischemia influenced telomere length. Q-FISH was validated against real-time PCR (the gold standard measure of telomere length) (Fig. 3). Fresh tissue from eight patients with liver disease obtained at liver resection or transplantation was analyzed in tandem using Q-FISH and real-time PCR. Cy5 staining of telomeres using Q-FISH (Fig. 1) provided data on telomere number, area, and intensity. Telomere intensity for all nuclei in liver tissue (not separated by cell lineage) correlated most closely with telomere length in liver homogenate measured by real-time PCR (R2 = 0.659, P = 0.015). Mean Cy5 intensity was therefore used in subsequent experiments as a measure of telomere length.