Application of Flow‐FISH for Dynamic Measurement of Telomere Length in Cell Division

Vyacheslav I. Borisov1, Olga Y. Korolkova1, Vladimir S. Kozhevnikov1

1 Research Institute of Clinical Immunology, Russian Academy of Medical Sciences, Novosibirsk
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 8.14
DOI:  10.1002/0471142956.cy0814s69
Online Posting Date:  July, 2014
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Abstract

This method makes it possible to measure the fluorescence of a DNA probe in cells with known division number and targeted surface antigen. In fact, this method is a combination or consistent application of three other methods: cell tracking by vital dye, surface immunophenotyping, and flow‐FISH. The idea in developing this method was to study telomere length changes in cells with known surface antigen after every new cell division. First, the in vitro cell culturing and staining with CFSE vital dye are performed. Then, cells are stained with surface MAbs labeled with biotin, followed by incubation with streptavidin‐labeled fluorochrome. After that, cells are fixed with BS3 reagent followed by the flow‐FISH procedure with PNA‐probe complementary to telomere DNA repeats. Finally, in one tube, it is possible to determine telomere length in surface antigen–labeled cells that have made the exact same number of divisions after incubation. Curr. Protoc. Cytom. 69:8.14.1‐8.14.10. © 2014 by John Wiley & Sons, Inc.

Keywords: telomeres; telomere length; flow‐FISH; cell tracker; CFSE

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Cell Division Number Measurement
  • Basic Protocol 2: Surface Immunophenotyping
  • Basic Protocol 3: Relative Telomere Length Measurement with Flow Fluorescence in Situ Hybridization (flow‐Fish)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Cell Division Number Measurement

  Materials
  • Cell suspension
  • RPMI 1640 medium, serum free (Sigma‐Aldrich, cat. no. R0883; store at 4°C)
  • 10 mM CFSE stock solution (see recipe)
  • Fetal bovine serum (FBS; Hyclone; store at 4°C)
  • Cell cultural medium (see recipe)
  • CO 2 incubator
  • 10‐ml centrifuge tubes
  • 25‐cm2 (60 ml) culture flasks

Basic Protocol 2: Surface Immunophenotyping

  Materials
  • Cell suspension after CFSE staining and cultivation ( protocol 1)
  • Anti‐CD4‐biotin monoclonal antibody (BD Pharmingen, cat. no. 555345); store at 4°C
  • Phosphate‐buffered saline (PBS; see recipe)
  • 50 μg/ml streptavidin‐Cy5 (see ; absorption = 650, emission = 670)
  • 25 mM BS3 [bis(sulfosuccinimidyl)suberate] stock solution (see recipe)
  • 1 М Tris·Cl, pH 7.1 ( appendix 2A)

Basic Protocol 3: Relative Telomere Length Measurement with Flow Fluorescence in Situ Hybridization (flow‐Fish)

  Materials
  • Cell suspension in PBS after surface immunophenotyping ( protocol 2)
  • Mouse splenocytes suspension (see recipe)
  • Hybridization solution (see recipe)
  • 30 μg/ml PNA‐(CCCTAA) 3‐Cy3 stock solution (peptide nucleic acid; absorption = 550; emission = 570; see recipe)
  • Washing solution 1 (see recipe)
  • Washing solution 2 (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 80°C heat block for 1.5‐ml microcentrifuge tubes
  • Shaker/rocker with 3‐D tumbling motion
  • Flow cytometer tubes
  • Flow cytometer
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Figures

Videos

Literature Cited

Literature Cited
  Baerlocher, G.M., Mak, J., Tien, T., and Lansdorp, P.M. 2002. Telomere length measurement by fluorescence in situ hybridization and flow cytometry: Tips and pitfalls. Cytometry 47:89‐99.
  Blackburn, E.H. 1991. Structure and function of telomeres. Nature 350:569‐573.
  Borisov, V.I., Korolkova, O.J., Blinova, E.V., Baev, D.A., Kozhevnikov, V.S., and Kozlov, V.A. 2012. Measuring telomere length in proliferating cells by flow‐FISH method. J. Life Sci. 6:945‐951.
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  Olovnikov, A.M. 1973. A theory of marginotomy. The incomplete copying of template margin in enzymic synthesis of polynucleotides and biological significance of the phenomenon. J. Theor. Biol. 41:181‐190.
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  Schmid, I., Dagarag, M.D., Hausner, M.A., Matud, J.L., Just, T., Effros, R.B., and Jamieson, B.D. 2002. Simultaneous flow cytometric analysis of two cell surface markers, telomere length, and DNA content. Cytometry 49:96‐105.
  Wright, W.E., Pereira‐Smith, O.M., and Shay, J.W. 1989. Reversible cellular senescence: Implications for immortalization of normal human diploid fibroblasts. Mol. Cell Biol. 9:3088‐3092.
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