Measurement of Intercellular Conjugates by Flow Cytometry

David M. Segal1

1 National Cancer Institute, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 5.6
DOI:  10.1002/0471142735.im0506s14
Online Posting Date:  May, 2001
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Abstract

Analysis of fluorescently labeled cellular aggregates can be accomplished quickly and accurately with the technique of flow cytometry. With a dual‐laser flow cytometer, the excitation and emission spectra of fluorophores used to label cells are widely separated, and there is no spillover of emission from one fluorophore into the detector measuring emission from the other. Consequently, conjugates can be measured by a dual‐laser instrument when the extent of labeling two cell types with fluorophores are very different. The more commonly available single‐laser cytometers can also be used to measure multicellular conjugates, but due to overlaps in emission spectra, the extent of labeling cells with fluorophores must be controlled much more carefully when the single‐laser machines are used. This unit describes the labeling of cells and analysis of conjugates with either dual‐laser or single laser flow cytometers.

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of Conjugates Using a Dual‐Laser Flow Cytometer
  • Alternate Protocol 1: Measurement of Conjugates Using a Single‐Laser Flow Cytometer
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Measurement of Conjugates Using a Dual‐Laser Flow Cytometer

  Materials
  • Phosphate‐buffered saline, pH 7.4 and 8.0 (PBS; appendix 2A)
  • 1 mM fluorescein isothiocyanate (FITC; e.g., Sigma), in 100% ethanol, freshly prepared
  • Complete RPMI‐10 medium ( appendix 2A)
  • Ficoll‐Hypaque solution (e.g., Pharmacia)
  • 1 mg/ml N‐hydroxysuccinimidobiotin (e.g., Sigma) in dimethyl sulfoxide (biotin/DMSO solution), freshly prepared
  • recipeTexas red–avidin solution (e.g., BRL)
  • recipeFlow cytometry (FC) medium
  • 15‐ml conical centrifuge tubes
  • Sorvall RC‐36 centrifuge with H‐2000B rotor (or equivalent)
  • 200‐µl microcentrifuge tubes
  • Dual‐laser flow cytometer with 488‐nm argon and 590‐nm dye lasers (e.g., Becton Dickinson or Coulter)
  • Additional reagents and equipment for preparing cells (unit 5.3), Ficoll‐Hypaque cell separation (unit 7.1), flow cytometry (unit 5.3), and analysis of data (unit 5.2)

Alternate Protocol 1: Measurement of Conjugates Using a Single‐Laser Flow Cytometer

  Additional Materials
  • recipePhycoerythrin (PE)‐avidin solution ( Becton Dickinson)
  • Single‐beam flow cytometer with a 488‐nm argon laser
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Figures

Videos

Literature Cited

Literature Cited
   Luce, G.G., Sharrow, S.O., and Shaw, S. 1985. Enumeration of cytotoxic cell‐target cell conjugates by flow cytometry using internal fluorescent stains. Bio Techniques 3:270‐272.
   Perez, P., Bluestone, J.A., Stephany, D.A., and Segal, D.M. 1985a. Quantitative measurements of the specificity and kinetics of conjugate formation between cloned cytotoxic T lymphocytes and splenic target cells by dual‐parameter flow cytometry. J. Immunol. 134:478‐485.
   Perez, P., Hoffman, R.W., Shaw, S., Bluestone, J.A., and Segal, D.M. 1985b. Specific targeting of cytotoxic T cells by anti‐T3 linked to anti‐target cell antibody. Nature (Lond.) 316:354‐356.
   Segal, D.M. 1988. The use of flow cytometry in the study of intercellular aggregation. In. Physical Basis of Cell‐Cell Adhesion (P. Bongrand, ed.) pp. 157‐172. CRC Press, Boca Raton, Fla.
   Segal, D.M. and Stephany, D.A. 1984a. The measurement of specific cell: cell interactions by dual‐parameter flow cytometry. Cytometry 5:169‐177.
   Segal, D.M. and Stephany, D.A. 1984b. The mechanism of intercellular aggregation. I. The kinetics of Fcγ receptor‐mediated aggregation of P388D1 cells with antibody‐coated lymphocytes at 4°C. J. Immunol. 132:1924‐1930.
Key Reference
   Segal, D.M. 1988. See above.
  A general review for measuring intercellular conjugates using flow cytometry.
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