Flow cytometric analysis of fluorescence in situ hybridization with dye dilution and DNA staining (flow‐FISH‐DDD) to determine telomere length dynamics in proliferating cells
- 19 October 2005
- journal article
- Published by Wiley in Cytometry Part A
- Vol. 68A (1), 53-58
- https://doi.org/10.1002/cyto.a.20181
Abstract
Background Telomeres shorten during DNA replication; extensive erosion of telomeres likely promotes replicative senescence and chromosomal instability. Telomere length in individual cells has been quantified by flow cytometric analysis of fluorescence in situ hybridization (flow‐FISH). To determine the rate of telomere attrition (telomere erosion per cell division), we combined flow‐FISH with dye dilution and DNA staining (flow‐FISH‐DDD) and measured telomere‐specific fluorescence in proliferating cells identified by cell generation and cell cycle phase. Methods Peripheral blood mononuclear cells (PBMC) were stained with the cell division tracking dye carboxyfluorescein diacetate succinimidyl ester (CFSE), stimulated with phytohemagglutinin (PHA), grown for 5–6 days, hybridized with a telomere sequence‐specific peptide nucleic acid fluorescent probe (PNA‐Cy5), counterstained with DAPI, and analyzed by flow cytometry. The cell cycle distribution and cell division generations were respectively identified by analysis of DAPI emission and deconvolution of CFSE emission, and Cy5 emission was used to determine telomere‐specific fluorescence, an indicator of telomere length, in each cell. Results In stimulated PBMC, in each cell cycle phase, the telomere‐specific fluorescence diminished with increasing cell generation. The rate of decline of the telomere‐specific fluorescence per cell generation did not significantly differ between cell cycle phases. Conclusions Application of flow‐FISH‐DDD to measure mean telomere length and the rate of telomere attrition in proliferating cells may find use in studies of ageing and disease, the effects of telomere‐modifying agents, and variability between individuals. © 2005 International Society for Analytical CytologyKeywords
This publication has 17 references indexed in Scilit:
- Telomere Length Measurements Using Fluorescence In Situ Hybridization and Flow CytometryMethods in Cell Biology, 2004
- Flow cytometric detection of accelerated telomere shortening in myelodysplastic syndromes: correlations with aetiological and clinical-biological findings*European Journal of Haematology, 2004
- Clonal haemopoiesis may occur after conventional chemotherapy and is associated with accelerated telomere shortening and defects in the NQO1 pathway; possible mechanisms leading to an increased risk of t‐AML/MDSBritish Journal of Haematology, 2004
- Recent advances in telomere biology: implications for human cancerCurrent Opinion in Oncology, 2004
- Association between telomere length in blood and mortality in people aged 60 years or olderThe Lancet, 2003
- Telomeres in T and B cellsNature Reviews Immunology, 2002
- Oxidative stress shortens telomeresTrends in Biochemical Sciences, 2002
- Replication Timing of Human Telomeric DNA and Other Repetitive Sequences Analyzed by Fluorescence in Situ Hybridization and Flow CytometryExperimental Cell Research, 2001
- Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometryNature Biotechnology, 1998
- A survey of telomerase activity in human cancerEuropean Journal Of Cancer, 1997