High‐Speed Cell Sorting

James F. Leary1

1 University of Texas Medical Branch, Galveston, Texas
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 1.7
DOI:  10.1002/0471142956.cy0107s01
Online Posting Date:  May, 2001
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A flow cytometer has the ability to analyze several thousand cells per second. This is particularly valuable for the detection of rare events. For many biological and clinical applications, cells must be isolated on the basis of multiple quantitative properties. This need is ideally met by the analytical power of flow cytometry, which provides the front‐end selection mechanism for cell sorting. In essence, cell sorting represents real‐time data classification coupled with actual physical isolation of cells with the properties of interest. This unit discusses the principles and practices of operating a cell sorter to maximize the sorting rate, including the trade‐offs between speed and resolution.

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Table of Contents

  • The New Importance of High‐Speed Cell Sorting
  • Some Important Aspects of Cell Sorting
  • The Effects of Instrument Deadtime on Cell‐Sorting Speeds
  • Theory and Practice of High‐Speed Cell Sorting
  • High‐Speed Sort Stability
  • Enrichment Strategies Employing High‐Speed Sorting
  • How to Calculate Sort Purity
  • Effects of Anti‐Coincidence on Sort Yields
  • Literature Cited
  • Figures
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Literature Cited

Literature Cited
   Corio, M.A. and Leary, J.F. April 1993. System for Flexibly Sorting Particles. U.S. patent 5,199,576.
   Gross, D. and Harris, C.M. 1985. Fundamentals of Queuing Theory, 2nd ed. John Wiley & Sons, New York.
   Herweijer, H., Stokdijk, W., and Visser, J.W. 1988. High‐speed photodamage cell selection using bromodeoxyuridine/Hoechst 33342 photosensitized cell killing. Cytometry 9:143‐149.
   Keij, J.F. 1994. The Zapper. Ph.D. dissertation. TNO Health Research Institute, The Netherlands.
   Keij, J.F., van Rotterdam, A., Groenewegen, A.C., Stokdijk, W., and Visser, J.W.M. 1991. Coincidence in high‐speed flow cytometry: Models and measurements. Cytometry 12:398‐404.
   Kompala, D.S. and Todd, P. (eds.) 1991. Cell separation science and technology. ACS Symposium Series no. 464. American Chemical Society, Washington, D.C.
   Leary, J.F. 1994. Strategies for rare cell detection and isolation. Methods Cell Biol. 42:331‐358.
   Leary, J.F., Corio, M.A., and McLaughlin, S.R. April 1993. System for High‐Speed Measurement and Sorting of Particles. U.S. patent 5,204,884.
   Leary, J.F., Ellis, S.P., McLaughlin, S.R., Corio, M.A., Hespelt, S., Gram, J.G., and Burde, S. 1991. High‐resolution separation of rare cell types. In Cell Separation Science and Technology, ACS Symposium Series no. 464 (D.S. Kompala and P. Todd, eds.) pp. 26‐40. American Chemical Society, Washington, D.C.
   Leary, J.F. and McLaughlin, S.R. 1995. New technology for ultrasensitive detection and isolation of rare cells for clinical diagnostics and therapeutics. In Progress in Biomedical Optics: Proceedings of Ultrasensitive Instrumentation for DNA Sequencing and Biochemical Diagnostics, Vol. 2386 (G.E. Cohn, J.M. Lerner, K.J. Liddane, A. Scheeline and S.A. Soper, eds.) pp. 150‐163. SPIE/Optical Society of America, Bellingham, Washington.
   Lindmo, T. and Fundingsrud, K. 1981. Measurements of the distribution of time intervals between cell passages in flow cytometry as a method for the evaluation of sample preparation procedures. Cytometry 2:151‐154.
   McCoy, J.P., Chambers, W.H., Lakomy, R., Campbell, J.A., and Stewart, C.C. 1991. Sorting minor subpopulations of cells: Use of fluorescence as the triggering signal. Cytometry 12:268‐274.
   Parson, J.D., Hiebert, R.D., and Martin, J.C. 1985. Active analog pipeline delays for high signal rates in multistation flow cytometers. Cytometry 6:388‐391.
   Peters, D., Branscomb, E., Dean, P., Merrill, T., Pinkel, D., Van Dilla, M., and Gray, J. 1985. The LLNL high‐speed sorter: Design features, operational characteristics, and biological utility. Cytometry 6:290‐301.
   van den Engh, G. and Stokdijk, W. 1989. Parallel processing data acquisition system for multilaser flow cytometry and cell sorting. Cytometry 10:282‐293.
   van Rotterdam, A., Keij, J., and Visser, J.W. 1992. Models for the electronic processing of flow cytometric data at high particle rates. Cytometry 13:149‐154.
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