Multiparameter FACS Analysis

Mario Roederer1

1 Vaccine Research Center, NIAID, NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 5.8
DOI:  10.1002/0471142735.im0508s49
Online Posting Date:  August, 2002
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The purpose of this unit is three‐fold: (1) toprovide a brief historical perspective to illustrate the demands for this technology and to lay the foundation for its application; (2) to explain the hurdles that were surmounted to achieve the current level of multiparametric analysis which serves to alert researchers to potential problems they may encounter when they either bring this technology to their own laboratories, or when they use extant instrumentation in another laboratory; and (3) to illustrate some of the complexities that arise even when all of the major technical hurdles have been overcome. The majority of the unit is focused on “high‐end” multiparameter analysis (termed “polychromatic flow cytometry,” or PFC); i.e., experiments utilizing more than four fluorescence detectors (in addition to scattered light parameters).

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Table of Contents

  • A Brief History of Polychromatic Flow Cytometry
  • Hurdles to Implementing PFC
  • The Power of PFC
  • Figures
  • Tables
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Literature Cited

Literature Cited
   Bagwell, C.B. and Adams, E.G. 1993. Fluorescence spectral overlap compensation for any number of flow cytometry parameters. Ann. N.Y. Acad. Sci. 677:167‐184.
   Baumgarth, N. and Roederer, M. 2000. A practical approach to multicolor flow cytometry for immunophenotyping. J. Immunol. Meth. 243:77‐97.
   Bigos, M., Baumgarth, N., Jager, G.C., Herman, O.C., Nozaki, T., Stovel, R.T., Parks, D.R., and Herzenberg, L.A. 1999. Nine color eleven parameter immunophenotyping using three laser flow cytometry. Cytometry 36:36‐45.
   De Rosa, S.C., Herzenberg, L.A., and Roederer, M. 2001. 11‐color, 13‐parameter flow cytometry: Identification of human naive T cells by phenotype, function, and T‐cell receptor diversity. Nat. Med. 7:245‐248.
   Lee, P.P., Yee, C., Savage, P.A., Fong, L., Brockstedt, D., Weber, J.S., Johnson, D., Swetter, S., Thompson, J., Greenberg, P.D., Roederer, M., and Davis, M.M. 1999. Characterization of circulating T cells specific for tumor‐associated antigens in melanoma patients. Nat. Med. 5:677‐685.
   Mitra, D.K., De Rosa, S.C., Luke, A., Balamurugan, A., Khaitan, B.K., Tung, J., Mehra, N.K., Terr, A.I., O'Garra, A., Herzenberg, L.A., and Roederer, M. 1999. Differential representations of memory T cell subsets are characteristic of polarized immunity in leprosy and atopic diseases. Intl. Immunol. 11:1801‐1810.
   Roederer, M. 1999. Compensation. In Current Protocols in Cytometry (J.P. Robinson, Z. Darzynkiewicz, P.N. Dean, L.G. Dressler, P.S. Rabinovitch, C.C. Stewart, H.J. Tanke, and L.L. Wheeless, eds.) pp. 1.14.1‐1.14.19. John Wiley & Sons, New York.
   Roederer, M. 2001 Spectral compensation for flow cytometry: Visualization artifacts, limitations, and caveats. Cytometry 45:194‐205.
   Roederer, M., DeRosa, S., Gerstein, R., Anderson, M., Bigos, M., Stovel, R., Nozaki, T., Parks, D., Herzenberg, L., and Herzenberg, L. 1997. 8‐Color, 10‐parameter flow cytometry to elucidate complex leukocyte heterogeneity. Cytometry 29:1‐12.
   Roederer, M., Kantor, A.B., Parks, D.R., and Herzenberg, L.A. 1996. Cy7PE and Cy7APC: Bright new probes for immunofluorescence. Cytometry 24:191‐197.
   Shapiro, H.M. 1994. Practical Flow Cytometry. Wiley‐Liss, New York.
   Stewart, C.C. and Stewart, S.J. 1999. Four color compensation. Cytometry 38:161‐175.
Internet Resources
  Compensation in flow cytometry: A perspective.
  Methods for fluorescent conjugation of monoclonal antibodies.
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