Centrifugal Elutriation to Obtain Synchronous Populations of Cells

Alan F. Wahl1, Karen L. Donaldson1

1 Seattle Genetics, Inc., Bothell, Washington
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 8.5
DOI:  10.1002/0471143030.cb0805s02
Online Posting Date:  May, 2001
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Abstract

Counterflow centrifugal elutriation is a noninvasive method for separating large numbers of cells on the basis of their size and mass. For mammalian cells, this method is useful for separating mixed populations of cells, in particular cells at different stages of the cell division cycle without perturbing cell metabolism or using synchronizing agents. This unit describes a method for separating 2 x 108 cells using the standard JE‐6B rotor or larger numbers of cells in the JE‐5.0 rotor. To verify the purity and to characterize the cell cycle positions of cells in the elutriated populations, the unit includes protocols for measuring nascent DNA synthesis by [3H]thymidine incorporation and for detecting DNA synthesis and content by propidium iodide flow cytometry alone or in combination with bromodeoxyuridine incorporation.

     
 
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Table of Contents

  • Basic Protocol 1: Separation of Cells into Progressive Stages of the Cell Division Cycle by Counterflow Centrifugal Elutriation
  • Alternate Protocol 1: Large‐Scale Elutriation with the Je‐5.0 Rotor
  • Analysis of Cell Fractions to Determine Cell Cycle Position
  • Support Protocol 1: Standard PI Staining and Flow Cytometric Analysis
  • Support Protocol 2: [3H]Thymidine Deoxyribose Incorporation
  • Support Protocol 3: Combined BrdU Incorporation and PI Staining
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Separation of Cells into Progressive Stages of the Cell Division Cycle by Counterflow Centrifugal Elutriation

  Materials
  • 70% (v/v) ethanol
  • Elutriation medium (see recipe) equilibrated ≥2 hr at ambient temperature
  • Nonadherent cells in log‐phase growth: suspension cultures at 2–5 × 105 cells/ml with fresh medium added at each doubling
  • Beckman J‐6B or J25I centrifuge with view port door and strobe assembly
  • Beckman JE‐6B rotor with standard elutriation and bypass chambers
  • Elutriation liquid system assembly (Fig. ), consisting of:
  •  Medium reservoir: 2‐liter roller bottle and 2‐ml pipet
  •  Loading syringe: 10‐ml syringe barrel and three‐way valve
  •  High‐quality peristaltic pump with 10‐turn potentiometer or digital control (e.g., Masterflex 7520 or 7550), capable of 100 rpm, 0 to 150 ml/min in 0.1 ml/min;increments
  •  Pulse/bubble trap and three‐way valve (Beckman)
  •  Pressure gauge (Beckman)
  •  Collection tube: 250‐ml conical centrifuge bottle (Corning)
  •  ¼‐in.‐o.d. (0.64‐cm) Silastic tubing (Masterflex 6411‐14)
  •  Ring stand and clamps
  • 18‐G needle
  • 10‐ml syringe
  • 250‐ml conical centrifuge bottles
  • Beckman GPR centrifuge (or equivalent) with swinging bucket rotor
  • Additional reagents and equipment for counting cells with a hemacytometer (unit 1.1)
NOTE: Elutriation is performed with the centrifuge, media, and cells at ambient temperature. Cell fractions are processed immediately after collection or stored on ice in multiple runs.

Alternate Protocol 1: Large‐Scale Elutriation with the Je‐5.0 Rotor

  • Beckman JE‐5.0 rotor with 40‐ml chamber
  • 3/8;‐in.‐o.d. (1.0‐cm) Silastic tubing (Masterflex 6411‐16)
  • 30‐ml syringes for loading chamber and for dispersing cells
  • 4‐liter vessel
  • Large sterile vessel
  • Large waste flask

Support Protocol 1: Standard PI Staining and Flow Cytometric Analysis

  Materials
  • Elutriated cell populations (see protocol 1 or protocol 2)
  • PBS ( appendix 2A), ice cold
  • Methanol, ice cold
  • Buffered PI solution (see recipe)
  • 200 U/ml RNase in PBS
  • 12 × 75–mm tubes
  • Nylon cloth, 41‐µm mesh
  • Flow cytometer (Becton Dickinson Immunocytometry FACScan or equivalent)
  • Additional reagents and equipment for counting cells with a hemacytometer (unit 1.1)

Support Protocol 2: [3H]Thymidine Deoxyribose Incorporation

  Materials
  • Elutriated cell populations (see protocol 1 or protocol 2)
  • Culture medium prewarmed to 37°C
  • [Methyl‐3H]thymidine deoxyribose ([3H]TdR; 6.7 Ci/mmol)
  • Aqueous liquid scintillation fluid
  • 96‐well microtiter plate
  • Glass fiber filters
  • Cell harvester

Support Protocol 3: Combined BrdU Incorporation and PI Staining

  Materials
  • Elutriated cell populations (see protocol 1 or protocol 2)
  • Culture medium
  • 1 mM BrdU solution (see recipe)
  • PTB buffer (see recipe)
  • PBS ( appendix 2A), ice cold
  • 100% ethanol, ice cold
  • 2 N HCl/0.5% (v/v) Triton X‐100 (store at 4°C)
  • 0.1 M sodium tetraborate (Na 2B 4O 7), pH 8.5 (store at 4°C)
  • Fluorescein isothiocyanate (FITC)–conjugated anti‐BrdU antibody
  • 5 µg/ml PI solution (see recipe)
  • 12 × 75–mm tubes
  • Flow cytometer (Becton Dickinson Immunocytometry FACScan or equivalent)
  • Additional reagents and equipment for counting cells with a hemacytometer (unit 1.1)
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Figures

Videos

Literature Cited

Literature Cited
   Beckman Instruments. 1981. The JE‐6B Elutriation System and Rotor Instruction Manual (JE6B‐IM‐2). The Spinco Division of Beckman Instruments, Palo Alto, Calif.
   Beckman Instruments. 1989. The JE‐5.0 Elutriation System Instruction Manual (JE5‐IM‐1). The Spinco Division of Beckman Instruments, Palo Alto, Calif.
   Donaldson, K.L., McShea, A., and Wahl, A.F. 1997. Separation by counterflow centrifugal elutriation and analysis of T‐ and B‐lymphocytic cell lines in progressive stages of cell division cycle. J. Immunol. Methods. 203:25‐33.
   Kauffman, M.G., Noga, S.J., Kelly, T.J., and Donnenberg, A.D. 1990. Isolation of cell cycle fractions by counterflow centrifugal elutriation. Anal. Biochem. 191:41‐46.
   Keng, P.C., Li, C.K.N., and Wheeler, K.T. 1981. Characterization of the separation properties of the Beckman elutriator system. Cell Biophys. 3:41‐56.
   Marjanovic, S., Skog, S., Heiden, T., Tribukait, B., and Nelson, B.D. 1991. Expression of glycolytic isoenzymes in activated human peripheral lymphocytes: Cell cycle analysis using flow cytometry. Exp. Cell Res. 193:425‐431.
   Meistrich, M. 1983. Cell separation: Methods and selected applications. In Experimental Factors Involved in Separation by Centrifugal Elutriation, Vol. 2 (T. Pretlow II and T. Pretlow, eds.) p.33. Academic Press, New York.
   Thompson, C.B., Ryan, J.J., Sieckmann, D.G., Finkelman, F.D., Mond, J.J., and Scher, I. 1983. A method for size separation of murine spleen cells using counterflow centrifugation. J. Immunol. Methods. 63:299‐307.
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