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

  •   FigureFigure 4.1.1 Cell culture synchronization. Two‐ or three‐day asynchronous lymphocyte cultures are blocked by addition of methotrexate, a folate antagonist that prevents thymidine synthesis. Depletion of the thymidine pool prevents the cells from completing replication, and cells accumulate in the synthetic, or S, phase of the cell cycle. Subsequent addition of thymidine releases a synchronized wave of cells to complete replication and proceed through G2 and into mitosis.
  •   FigureFigure 4.1.2 A partial karyotype of G‐banded chromosome 7 from cells at successive stages of mitosis, illustrating the coalescing of subbands into larger, less defined, and less informative landmark bands. Cells captured in late prophase may show ≥850 bands per haploid karyotype, i.e., high resolution. By mid‐metaphase, fine band detail is lost as chromosomes condense and bands fuse, and only 400 or fewer bands per haploid karyotype may be observed.
  •   FigureFigure 4.1.3 High‐resolution harvest. Prometaphase chromosomes are obtained by sequential harvests of synchronized cultures (1, 2, and 3), timed to capture a wave of cells early in mitosis, when the chromosomes are longest. Staggered 10‐min mitotic arrests span a 30‐min harvest window. The short mitotic arrest prevents excessive chromosome condensation. Addition of ethidium bromide ∼1 hr before mitotic arrest directly inhibits chromosome condensation. In a typical high‐resolution harvest, culture 1 may yield little or no mitoses, culture 2 may capture an early mitotic wave with long chromosomes, and culture 3 mitoses are shorter, having proceeded further into metaphase.
  •   FigureFigure 4.1.4 Chromosome slide preparation: (A) After blotting the long edge of the slide to obtain a thin uniform layer of water, the slide is tilted to ∼30° and 3 separate drops of fixed cell suspension are applied starting away from and proceeding toward the frosted end. This sequence allows excess fixative and water to flood onto the frosted end without pooling on the slide. Application of the drops 1/3 of the distance from the top of the slide (indicated by Xs) counteracts the downhill dispersal tendency of cells on the slide and promotes even dispersal across the slide width. (B) After application of the cell suspension, the slide is flooded with fixative across the top edge, again proceeding toward the frosted end. This displaces a front of remaining water across the slide and onto the frosted end. It is important to avoid pooling of excess fluid on the surface of the slide, and to obtain a thin, even film of fixative to ensure uniform drying.
  •   FigureFigure 4.1.5 (A) A G‐banded high resolution metaphase. Phase‐contrast photomicrographs show (B) low‐power field (16× objective) with many polysegmented nuclei, small nuclei, and a lack of metaphases associated with failure of PHA mitogenic stimulation; (C) poor spreading of “sticky” chromosomes from two mitoses caused by excessive ethidium bromide exposure; (D) low‐power field showing an appropriate and moderate density of cells with two nicely spread metaphases; (E) low‐power field showing excessive cell density and resultant poor spreading of two metaphases; and (F) category IV metaphase of poorly spread refractile chromosomes with encapsulation in cellular debris due to improper regulation of the slide‐drying regimen.
  •   FigureFigure 4.1.6 Sequential phase contrast and G‐band photomicrographs of the different categories of metaphase. Category I metaphases (A, B) have a gray, fuzzy phase‐contrast appearance and yield ragged, poor‐quality G‐bands. Categories II (C, D) and III (E, F) show increasingly sharp and darkly defined phase contrast and are optimal for in situ hybridization and G‐banding, respectively. Category IV metaphases (G, H) are refractile in appearance with excessive phase contrast. They yield G‐bands with high contrast and a loss of subtle detail and are unable to withstand the in situ hybridization denaturation process unless aged.

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Literature Cited

Literature Cited
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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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