Ten‐Color Immunophenotyping of Hematopoietic Cells

Brent L. Wood1

1 University of Washington, Seattle, Washington
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.21
DOI:  10.1002/0471142956.cy0621s33
Online Posting Date:  August, 2005
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Abstract

Multicolor flow cytometry offers the unique ability to simultaneously assess and correlate multiple cellular properties at the single‐cell level in a timely and efficient manner. Principles necessary for the development and evaluation of 10‐color flow cytometry panels are discussed. The Basic Protocol outlines a simple and efficient method for the labeling of white blood cells with monoclonal antibodies directed against cell surface antigens. Alternate Protocol 1 incorporates the removal of plasma to allow the simultaneous assessment of surface light‐chain expression on B cell populations. Alternate Protocol 2 describes a general method for the simultaneous assessment of surface and cytoplasmic antigens using a combination of fixation followed by membrane permeabilization. The methods were developed in a clinical laboratory setting for the description of normal pathways of hematopoietic maturation and the efficient identification of neoplastic hematopoietic cell populations, but the general principles should also be suitable for other applications.

Keywords: immunophenotyping; multicolor; hematopoietic cells; flow cytometry; antibody panel design

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Simultaneous Labeling of White Blood Cells with Multiple Monoclonal Antibodies
  • Alternate Protocol 1: Removal of Plasma for the Simultaneous Labeling of White Blood Cells with Multiple Monoclonal Antibodies
  • Alternate Protocol 2: Simultaneous Labeling of White Blood Cells with Multiple Monoclonal Antibodies To Surface and Cytoplasmic Antigens
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Simultaneous Labeling of White Blood Cells with Multiple Monoclonal Antibodies

  Materials
  • Antibody cocktail (see and Table 6.21.1)
  • Cell suspension (white cell concentration <10,000 cells/µl)
  • Buffered NH 4Cl lysing solution containing 0.25% formaldehyde (see recipe)
  • PBS/BSA/azide (see recipe)
  • 12 × 75–mm polystyrene tubes
  • Benchtop centrifuge
  • Multilaser flow cytometer with appropriate filters for collection of fluorescence emission

Alternate Protocol 1: Removal of Plasma for the Simultaneous Labeling of White Blood Cells with Multiple Monoclonal Antibodies

  • RPMI medium containing 10% newborn calf serum (NBCS; see recipe)
  • Light‐chain antibody cocktail, titered (see and Table 6.21.1)

Alternate Protocol 2: Simultaneous Labeling of White Blood Cells with Multiple Monoclonal Antibodies To Surface and Cytoplasmic Antigens

  • Surface antibody cocktail, titered (see and Table 6.21.1)
  • Fix and Perm kit (Caltag Laboratories; alternatives include Intrastain from Dako/Cytomation or Intraprep from Beckman Coulter)
  • Cytoplasmic antibody cocktail, titered (see and Table 6.21.1)
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Figures

Videos

Literature Cited

Literature Cited
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   Berhanu, D., Mortari, F., De Rosa, S.C., and Roederer, M. 2003. Optimized lymphocyte isolation methods for analysis of chemokine receptor expression. J. Immunol. Methods 279:199‐207.
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   Renzi, P., and Ginns, L.C. 1987. Analysis of T cell subsets in normal adults: Comparison of whole blood lysis technique to Ficoll‐Hypaque separation by flow cytometry. J. Immunol. Methods 98:53‐56.
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   Romeu, M.A., Mestre, M., Gonzalez, L., Valls, A., Verdaguer, J., Corominas, M., Bas, J., Massip, E., and Buendia, E. 1992. Lymphocyte immunophenotyping by flow cytometry in normal adults: Comparison of fresh whole blood lysis technique, Ficoll‐Paque separation and cryopreservation. J. Immunol. Methods 154:7‐10.
   Wood, B.L. 2004. Multicolor immunophenotyping: Human immune system hematopoiesis. Methods Cell Biol. 75:559‐576.
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