Metaphase Chromosome Preparation from Cultured Peripheral Blood Cells

Charles D. Bangs1, Timothy A. Donlon2

1 Stanford University Hospital, Stanford, California, 2 The Queen's Medical Center, Honolulu, Hawaii
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 4.1
DOI:  10.1002/0471142905.hg0401s45
Online Posting Date:  May, 2005
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Abstract

Chromosome preparations currently provide the only direct view of the genome as a whole. Although molecular methods allow a more detailed analysis of specific regions of the genome, the study of genetics is not complete without an appreciation of the metaphase cell. The stimulated T cell system described in this unit is the most widely used means of obtaining large numbers of mitotic cells for genetic analyses. Synchronization of the cell cycle in culture is described, combined with direct inhibition of chromosome condensation, to yield longer high‐resolution prophase or prometaphase preparations. Such preparations are used for detailed analysis of microdeletions or subtle rearrangements, fine breakpoint analysis, and refined mapping. Microscope slide preparation of mitotic chromosomes from harvested cell culture suspensions is also explained in the support protocol.

     
 
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Table of Contents

  • Basic Protocol 1: Culture and Metaphase Harvest of Peripheral Blood
  • Alternate Protocol 1: Culture and Harvest for High‐Resolution Prometaphase Chromosomes
  • Support Protocol 1: Chromosome Slide Preparation
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Culture and Metaphase Harvest of Peripheral Blood

  Materials
  • Heparinized whole blood obtained via Vacutainer (Becton Dickinson) or syringe with preservative‐free sodium heparin (25 U/ml)
  • Complete RPMI/10% FBS medium ( appendix 3G) containing 50 µg/ml gentamycin sulfate in place of penicillin and streptomycin
  • 100× phytohemagglutinin‐M (PHA) stock (GIBCO/BRL), reconstituted in sterile deionized water (store at 4°C)
  • 10 µM methotrexate (optional; see recipe)
  • 1 mM thymidine (optional; see recipe)
  • 10 µg/ml Colcemid (GIBCO/BRL)
  • 75 mM KCl (0.56 g in 100 ml H 2O; store ≤2 weeks at room temperature)
  • Fixative: 3:1 (v/v) HPLC‐grade absolute methanol/glacial acetic acid, (prepare fresh)
  • 15‐ml sterile disposable conical polypropylene centrifuge tubes
  • TB syringe equipped with 21‐G needle (VWR Scientific)
  • IEC HN‐SII centrifuge with 958 rotor (or equivalent)
CAUTION: Human blood and methotrexate are hazardous; see appendix 2A for guidelines on handling storage, and disposal.NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Alternate Protocol 1: Culture and Harvest for High‐Resolution Prometaphase Chromosomes

  • 1.25 mM ethidium bromide solution (see recipe)
CAUTION: Ethidium bromide is hazardous; see appendix 2A for guidelines on handling, storage, and disposal. NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 1: Chromosome Slide Preparation

  Materials
  • Fixed cultures (basic or alternate protocols)
  • Fixative: 3:1 (v/v) methanol/acetic acid (use 100% methanol and glacial acetic acid, both AR grade, from J.T. Baker)
  • Microscope slides (one end frosted) stored in 100% methanol (absolute AR grade, J.T. Baker) in Coplin jars
  • Lint‐free tissue (e.g., Kimwipe or gauze pad)
  • Zeiss Standard phase‐contrast microscope with 16× Ph2 objective and condenser ring (or equivalent)
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Figures

Videos

Literature Cited

Literature Cited
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   Barch, M.J. (ed.) 1991. The ACT Cytogenetics Laboratory Manual, 2nd ed. Raven Pressv, New York.
   Barch, M.J., Lawce, H.J., and Arsham, M.S. 1991. Peripheral Blood Culture. In The ACT Cytogenetics Laboratory Manual, 2nd ed. (M.J. Barch, ed.) pp. 17‐30. Raven Press, New York.
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   Lawce, H.J. and Brown, M.G. 1991. Harvesting, slide‐making and chromosome elongation techniques. In The ACT Cytogenetics Laboratory Manual, 2nd ed. (M.J. Barsch ed.) pp. 31‐66. Raven Press, New York.
   Ledbetter, S.A., Kuwano, A., Dobyns, W.B., and Ledbetter, D.H. 1992. Microdeletions of chromosome 17p13 as a cause of isolated lissencephaly. J. Hum. Genet. 50:182‐189.
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Key References
   Barch, M.J., 1991. See above.
  Comprehensive treatment of the full range of cytogenetic technology, with an in‐depth discussion of chromosome harvest and slide preparation by the Association of Cytogenetic Technologists.
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