Practical Issues in High‐Speed Cell Sorting

Larry W. Arnold1, Joanne Lannigan2

1 The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 2 The University of Virginia, Charlottesville, Virginia
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 1.24
DOI:  10.1002/0471142956.cy0124s51
Online Posting Date:  January, 2010
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Modern flow cytometric cell sorters are all capable of so‐called “high‐speed sorting.” However, there is confusion about exactly how fast a “high‐speed” cell sorter can sort cells. There are many considerations in achieving the fastest sorting speed, as well as the highest quality sort results—cell recovery, purity, and functionality. This requires the same considerations required for “slow‐speed sorting”; however, a more precise implementation is required for high‐speed sorting. The modern cell sorters enable high‐speed sorting because of advances in high‐speed electronics and data processing. We discuss the practical considerations of high‐speed sorting in terms of the theory and practical aspects of the mechanical and software components of sorting, statistics of sorting, cell preparation and viability, instrument setup, sort strategies, and biosafety. Curr. Protoc. Cytom. 51:1.24.1‐1.24.30. © 2010 by John Wiley & Sons, Inc.

Keywords: flow cytometry; sorting; FACS

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • What is High‐Speed Sorting?
  • Understanding High‐Speed Sorting
  • Practical Aspects of High‐Speed Sorting
  • Safety
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Alberti, S., Parks, D.R., and Herzenberg, L.A. 1987. A single laser method for subtraction of cell autofluorescence in flow cytometry. Cytometry 8:114‐119.
   Lennartz, K., Lu, M., Flasshove, M., Moritz, T., and Kirsten, U. 2005. Improving the biosafety of cell sorting by adaptation of a cell sorting system to a biosafety cabinet. Cytometry 66:119‐127.
   Maecker, H.T., Frey, T., Nomura, L.E., and Trotter, J. 2004. Selecting fluorochromes for maximum sensitivity. Cytometry 62:169‐173.
   Oberyszyn, A.S. and Robertson, F.M. 2001. Novel rapid method for visualization of extent and location of contamination during high‐speed sorting of potential biohazardous samples. Cytometry 43:217‐222.
   Perfetto, S.P., Ambrozak, D.R., Koup, R.A., and Roederer, M. 2003. Measuring containment of viable infectious cell sorting in high‐velocity cell sorters. Cytometry 52:122‐130.
   Perfetto, S.P., Ambrozak, D.R., Roederer, D.R., and Koup, R.A. 2004. Viable infectious cell sorting in a BSL‐3 facility. Methods Cell Biol. 263:419‐424.
   Peters, D., Branscomb, E., Dean, P., Merrill, T., Pinkel, D., Van Dilla, M., and Gray, J.W. 1985. The LLNL high‐speed sorter: Design features, operational characteristics, and biological utility. Cytometry 6:290‐301.
   Pinkel, D. and Stovel, R. 1985. Flow chambers and sample handling. In Flow Cytometry: Instrumentation and Data Analysis (P.N. Dean, ed.) pp. 78‐128. Academic Press, London.
   Rayleigh, J.W.S. 1877. Theory of Sound. MacMillan, London. Reprinted 1945. Dover, New York.
   Roederer, M. and Murphy, R.F. 1986. Cell‐by‐cell autofluorescence correction for low signal‐to‐noise systems: Applications to epidermal growth factor endocytosis by 3T3 fibroblasts. Cytometry 7:558‐565.
   Schmid, I., Nicholson, J.K.A., Giorgi, J.V., Janossy, G., Kunkl, A., Lopez, P.A., Perfetto, S., Seamer, L.C., and Dean, P.N. 1997. Biosafety guidelines for sorting unfixed cells. Cytometry 28:99‐117.
   Schmid, I., Lambert, C., Ambrozak, D., Marti, G.E., Moss, D.M., and Perfetto, S.P. 2007. International society for analytical cytology biosafety for sorting of unfixed cells. Cytometry 71:414‐437.
   Van den Engh, G. 2000. High speed cell sorting. In Emerging Tools for Single‐Cell Analysis (G. Durack and J.P. Robinson, eds.) pp. 21‐48. Wiley‐Liss, New York.
   van den Engh, G.J. and Stokdijk, W. 1989. Parallel processing data acquisition system for multi‐laser flow cytometry and cell sorting. Cytometry 10:282‐293.
   Van Dilla, M.A., Deaven, L.L., Albright, K.L., Bartholdi, N.C., Campbell, E.W., Carrano, A.V., Christensen, M., Clark, L.M., Cram, L.S., Dean, P.N., de Jong, P., Fawcett, J.J., Fuscoe, J.C., Gray, J.W., Hildebrand, C.E., Jackson, P.J., Jett, J.H., Killa, S., Longmire, J.L., Lozes, C.R., Luedemann, M.L., McNinch, J.S., Mendelsohn, M.L., Meyne, J., Meincke, L.J., Moyzis, R.K., Mullikin, J., Munk, A.C., Perlman, J., Pederson, L., Peters, D.C., Silva, A.J., Trask, B.J., van den Engh, G. 1990. The national laboratory gene library project. In Flow Cytogenetics (J.W. Gray, ed.) pp. 257‐274. Academic Press, London.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library