Assessment of Telomere Length, Phenotype, and DNA Content

Theodoros Kelesidis1, Ingrid Schmid2

1 Department of Medicine, Division of Infectious Diseases, David Geffen School of Medicine at UCLA, Los Angeles, California, 2 Department of Medicine, Hematology/Oncology, David Geffen School of Medicine at UCLA, Los Angeles, California
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.26
DOI:  10.1002/cpcy.12
Online Posting Date:  January, 2017
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Abstract

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 G0/1 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. © 2017 by John Wiley & Sons, Inc.

Keywords: fluorescence in situ hybridization; Flow‐FISH; telomeres; telomere length; cell surface immunofluorescence

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Analysis of Immunophenotype, Telomere Length, and DNA Content
  • Support Protocol 1: DNA Ploidy Determination
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Analysis of Immunophenotype, Telomere Length, and DNA Content

  Materials
  • PBS‐washed cells
  • Staining solution (see recipe)
  • PBS ( appendix 2A), room temperature and 40°C
  • Cross‐linking solution (see recipe)
  • 1 M Tris·Cl, pH 8.0 ( appendix 2A)
  • Internal control cells, cryopreserved (see recipe)
  • Hybridization buffer (see recipe) with and without fluorescently labeled, telomere‐specific PNA probe, (C 3TA 2) 3 (PerSeptive Biosystems or equivalent)
  • Nucleic‐acid stain compatible with other fluorochromes used (e.g., PI, 7‐AAD, Hoechst 33342), in PBS
  • 12 × 75–mm polystyrene tubes
  • 1.5‐ml polypropylene tubes
  • 80°C shaking water bath
  • 40°C water bath or heating block
  • Flow cytometer with 488‐nm blue excitation, 633‐nm red excitation, and optional UV or violet excitation depending on fluorochrome choices made during strategic planning of the experiments
  • Additional reagents and equipment for staining cells (unit 6.2; Stewart and Stewart, ), counting cells ( appendix 3A; Phelan and Lawler, ), and standardizing flow cytometers (unit 1.3; Hoffman, )

Support Protocol 1: DNA Ploidy Determination

  Materials
  • Unstained cells
  • Thawed internal control cells
  • PBS ( appendix 2A)
  • PI staining solution
   
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Figures

Videos

Literature Cited

   
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Key Reference
  Schmid et al., 2002. See above.
  Describes the application of the procedure presented in the Basic Protocol to the simultaneous measurement of two cell surface markers and telomere length.
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