Telomere length and telomerase activity. Human ECFCs derived from PB (PB ECFCs; n = 4) and umbilical cord blood (UCB ECFC; n = 4) after different culture passages (p1-p3) were compared with each other and with mouse 3T3 fibroblasts as a positive control. (A-D) Flow cytometry fluorescence in situ hybridization was used to determine telomere length and (E) TRAP to measure telomerase activity. (A,B) Gating strategy of one representative example to select single cells in cell cycle phase G_0/1 based on size parameters (FSC-H indicates forward light scatter height; SSC-H, sideward light scatter height; region R1 = 88.6%) and DNA content determined after propidium iodine (PI) staining of hybridized cells (PI fluorescence width vs area, FL2-W, FL2-A; region R2 = 77.7% of region R1). (C) Corresponding histogram showing fluorescein isothiocyanate-tagged peptide nucleic acid (PNA-FITC) binding to telomeres (gray histogram) compared with background fluorescence after mock hybridization (open histogram). (D) Differences between PNA probe specific signal and background fluorescence height (ΔFL1-H; mean ± SD, n = 4 per passage) based on analyses of at least 10 000 single cells in G_0/1 phase per sample. (E) Telomerase activity displayed as cycle of threshold (Ct) in a real-time polymerase chain reaction-based TRAP assay. Statistically significant differences: *P < .05, **P < .01.