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.
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.
Potter
Alan J
AJ
Immunology Division, Department of Laboratory Medicine, University of Washington, Seattle, Washington 98195, USA.
Wener
Mark H
MH
D002232:Carbocyanines / Q000737:chemistry; D002453:Cell Cycle / Q000502:physiology; D049109:Cell Proliferation; D016922:Cellular Senescence / Q000235:genetics; D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007211:Indoles / Q000737:chemistry; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D020135:Peptide Nucleic Acids / Q000737:chemistry; D010835:Phytohemagglutinins / Q000494:pharmacology; D013194:Staining and Labeling / Q000379:methods; D013388:Succinimides / Q000737:chemistry; D016615:Telomere / Q000378:metabolism*
C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D002232:Carbocyanines; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007211:Indoles; D020135:Peptide Nucleic Acids; D010835:Phytohemagglutinins; D013388:Succinimides; C085321:cyanine dye 5; C007293:DAPI; D004247:DNA