Assessment of telomere length, phenotype, and DNA content.
Telomere sequences at the end of chromosomes control somatic cell division; therefore, telomere length in a given cell population provides information about its replication potential. This unit describes a method for flow cytometric measurement of telomere length in subpopulations using fluorescence in situ hybridization of fluorescently-labeled probes (Flow-FISH) without prior cell separation. After cells are stained for surface immunofluorescence, antigen-antibody complexes are covalently cross-linked onto cell membranes before FISH with a telomere-specific probe. Cells with long telomeres are included as internal standards. Addition of a DNA dye permits exclusion of proliferating cells during data analysis. DNA ploidy measurements of cells of interest and internal standard are performed on separate aliquots in parallel to Flow-FISH. Telomere fluorescence of G(0/1) cells of subpopulations and internal standards obtained from Flow-FISH are normalized for DNA ploidy and telomere length in subsets of interest is expressed as a fraction of the internal standard telomere length.
Schmid
Ingrid
I
David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA.
Jamieson
Beth D
BD
D000818:Animals; D004247:DNA / Q000032:analysis*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D010641:Phenotype; D011003:Ploidies; D016192:Resting Phase, Cell Cycle; D016615:Telomere / Q000737:chemistry* / Q000648:ultrastructure
D004247:DNA
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