Preparation of Cytogenetic Specimens from Tissue Samples

Jane Bayani1, Jeremy A. Squire1

1 University of Toronto, Ontario
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 22.2
DOI:  10.1002/0471143030.cb2202s23
Online Posting Date:  September, 2004
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Abstract

Cytogenetic specimens are prepared from short‐term primary cultures established from tissue samples or from established cell lines. Cultures that are (80% confluent are treated with Colcemid to enrich for cells in mitosis. Cells are then swollen hypotonically and fixed before slides are prepared. Preparing good metaphase spreads is technically challenging, This unit provides a good discussion of the factors that go into making a good preparation.

Keywords: cell culture; cytogenetic preparations; metaphase spreads; tissue samples

     
 
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Table of Contents

  • Basic Protocol 1: Culturing Cells Derived from Solid Tissues for Chromosomal Analysis
  • Alternate Protocol 1: Culturing Tissues in Situ
  • Support Protocol 1: Prepare Glass Coverslips or Glass Slides for in Situ Culturing
  • Alternate Protocol 2: Culturing Nonadherent or Fluid Tissues for Chromosomal Analysis
  • Alternate Protocol 3: Culture of Peripheral Blood Cells
  • Basic Protocol 2: Preparation of Metaphase Spreads
  • Alternate Protocol 4: Cytogenetic Preparations from in Situ Cultures
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Culturing Cells Derived from Solid Tissues for Chromosomal Analysis

  Materials
  • Solid tissue specimen maintained in transfer medium
  • Appropriate culture medium (unit 1.2media for culture of mammalian cells)
  • 10× collagenase IV stock (see recipe)
  • Sterile medium‐sized petri dishes (e.g., 60‐mm)
  • Tabletop centrifuge
  • Scalpel and scalpel blades
  • Vented 25‐cm2 tissue culture flasks or 10‐cm diameter petri dishes
  • 15‐ml conical centrifuge tube
  • Additional reagents and equipment for cell culture (Chapter 1)
NOTE: All tissue culture incubations are performed in a humidified 37°C, 5% CO 2 incubator. Some media, e.g., DMEM, require increased levels of CO 2 to maintain the medium at pH 7.4.NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper aseptic technique must be used.

Alternate Protocol 1: Culturing Tissues in Situ

  • Poly‐L‐lysine‐coated glass coverslips or slides (see protocol 3) or chambered glass tissue culture slides (Nunc)
  • Phase‐contrast microscope
NOTE: All tissue culture incubations are performed in a humidified 37°C, 5% CO 2 incubator. Some media, e.g., DMEM, require increased levels of CO 2 to maintain the medium at pH 7.4.NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper aseptic technique must be used.

Support Protocol 1: Prepare Glass Coverslips or Glass Slides for in Situ Culturing

  Materials
  • Glass coverslips or slides, autoclaved
  • 1 mg/ml poly‐L‐lysine stock solution: mix 100 mg poly‐L‐lysine (Sigma) with 100 ml sterile H 2O
  • Sterile petri dishes or Coplin jar

Alternate Protocol 2: Culturing Nonadherent or Fluid Tissues for Chromosomal Analysis

  • Fluid specimen maintained in transfer medium
NOTE: All tissue culture incubations are performed in a humidified 37°C, 5% CO 2 incubator. Some media, e.g., DMEM, require increased levels of CO 2 to maintain the medium at pH 7.4.NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper aseptic technique must be used.

Alternate Protocol 3: Culture of Peripheral Blood Cells

  Materials
  • Blood collected in heparin
  • Phosphate‐buffered saline (PBS; appendix 2A), sterile
  • Ficoll‐Hypaque (density 1.077; Amersham Biosciences; store at 4°C protected from direct light)
  • Supplemented RPMI 1640 medium (see recipe)
  • 50‐ml conical tubes (e.g., Falcon)
  • 15‐ml conical centrifuge tubes (e.g., Falcon)
  • Refrigerated centrifuge
  • Sterile disposable plastic transfer pipets (Fisher)
  • 25‐cm2 tissue culture flasks
NOTE: All tissue culture incubations are performed in a humidified 37°C, 5% CO 2 incubator. Some media, e.g., DMEM, require increased levels of CO 2 to maintain the medium at pH 7.4.NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper aseptic technique must be used.

Basic Protocol 2: Preparation of Metaphase Spreads

  Materials
  • Adherent or nonadherent cell culture ( protocol 1 or protocol 4 or protocol 5), 80% confluent
  • 10 µg/ml Colcemid (Invitrogen) in H 2O (store up to 2 months at 4°C)
  • Appropriate tissue culture medium with serum
  • 1× trypsin/citrate saline (see recipe)
  • 0.075 M KCl (see recipe), prewarmed to 37°C in oven or water bath
  • 3:1 (v/v) methanol/acetic acid fixative (see recipe)
  • 15‐ml conical centrifuge tubes
  • Tabletop centrifuge
  • Sterile disposable plastic transfer pipets (Fisher)
  • Coplin jars
  • Glass slides
  • Slide box
  • Additional reagents and equipment for cell culture (Chapter 1), including trypsinization (unit 1.1basic techniques in mammalian cell tissue culture)

Alternate Protocol 4: Cytogenetic Preparations from in Situ Cultures

  • In situ cultures ( protocol 2), 80% confluent
  • Cytoseal 60 mounting medium (Richard‐Allan Scientific)
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Figures

Videos

Literature Cited

   Barch, M.J. 1991. The ACT Cytogenetics Laboratory Manual, 2nd Edition. Raven Press, New York.
   Freshney, R.R. 1993. Culture of Animal Cells: A Manual of Basic Techniuqes, 3rd Ed. Wiley‐Liss, New York.
   Henegariu, O., Heerema, N.A., Wright, L.L., Bray‐Ward, P., Ward, D.C., and Vance, G.H. 2001. Improvements in cytogenetic slide preparation: Controlled chromosome spreading, chemical aging and gradual denaturing. Cytometry. 43:101‐109.
   Hungerford, D., Donnely, A., Nowel, A., and Beck, S. 1959. The chromosome constitution of a human phenotypic intersex. Am. J. Hum. Genet. 11:215.
   Moorhead, P., Nowell, P., Mellman, W., Battips, D., and Hungerford, D. 1960. Chromosome preparations of leukocytes cultured from human peripheral blood. Exp.l Cell Res. 20:613.
   Rothfels, K. and Siminovitch, L. 1958. An air‐drying technique for flattening mammalian cells grown in vitro. Stain. Technol. 33:73.
   Squire, J. 1983. Dilution of culture medium of trypsinized cells as an alernative to conventional KCl hypotonic treatment. Karyogram. 9:27
Internet Resources
   http://www.thermotron.com/cryogen.html
  Thermotron Web site for equipment specifications and description.
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