Comparison of telomere analysis by flow-FISH technique and Southern blotting.

A total of 14 PBMC samples were collected from individuals of different ages ranging from umbilical cord blood samples to individuals over 70 years old. Cells were cyropreserved in 10% DMSO for telomere length analysis by flow-FISH and snap frozen as pellets for Southern blotting. Cells analyzed for telomere length by flow-FISH were all processed and run on the same day and all samples for Southern blot analysis were also run simultaneously (A). Purified CD45RA⁺and CD45RO⁺ T lymphocytes were purified using the MACS separation protocols (see “Patients, materials, and methods”) and then processed for flow-FISH as described (B). The histogram profiles show CD45RA⁺ T cells (open histogram) and CD45RO⁺ T cells (closed histogram). Dotted histogram represents the control X-chromosome–FITC probe. Comparison of telomere length using the flow-FISH of umbilical cord blood PBMCs and PBMCs from an individual over 60 years of age is shown in panel C. The histogram shows cord blood PBMCs (closed histogram) versus PBMCs from an over 60 year old (open histogram). Dotted histogram represents the control PNA probe (X-chromosome–FITC). All cells analyzed for flow-FISH were gated on live, nonblastoid cells as described (Figure 1B). At least 50 000 live events were acquired.