Immunophenotypic Identification, Enumeration, and Characterization of Human Peripheral Blood Dendritic Cells and Dendritic‐Cell Precursors

Julia Almeida1, Clara Bueno1

1 Universidad de Salamanca, Salamanca
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.9
DOI:  10.1002/0471142956.cy0609s17
Online Posting Date:  August, 2001
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Abstract

Study of dendritic cells is complicated by their low frequency and the lack of specific markers for sensitive and specific identification. Furthermore, these cells are not a homogeneous population, but contain a variety of subsets. Flow cytometry has well‐established applications for the analysis of cells present at low frequencies; the use of a two‐step acquisition procedure increases the number of cells for analysis. This unit presents protocols for the identification and enumeration of dendritic cells and cell subsets in erythrocyte‐lysed whole peripheral blood. Special attention is paid to the combinations of reagents used for the identification of all dendritic cells as well as each subset, and on the multiparametric gating strategies for specific analysis.

     
 
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Table of Contents

  • Basic Protocol 1: Immunophenotypic Identification and Enumeration of Peripheral Blood Dendritic Cells by Flow Cytometry
  • Alternate Protocol 1: Flow Cytometric Enumeration of PB Dendritic Cells Using Microbeads as an Internal Reference to Derive Absolute Cell Counts
  • Support Protocol 1: Immunophenotypic Characterization of PB Dendritic Cells by Flow Cytometry
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Immunophenotypic Identification and Enumeration of Peripheral Blood Dendritic Cells by Flow Cytometry

  Materials
  • Whole anticoagulated peripheral blood (PB) containing ∼1.5 × 106 nucleated cells
  • Fluorochrome‐conjugated monoclonal antibody mixture (unit 4.2):
  •  Fluorescein isothiocyanate (FITC)‐conjugated anti‐CD3, ‐CD19, ‐CD56, and ‐CD14
  •  Phycoerithrin (PE)‐conjugated anti‐CD16
  •  Peridinin chlorophyll protein (PerCP)‐, PE‐cyanine 5 (PE‐Cy5)‐ or PerCP‐cyanine 5.5 (PerCP‐Cy5.5)‐conjugated anti‐HLA‐DR
  •  Allophycocyanin (APC)‐ or PE‐Texas Red‐conjugated anti‐CD33
  • 1× ammonium chloride lysing solution ( appendix 2A), freshly prepared
  • Phosphate buffered saline (PBS; appendix 2A)
  • PBS containing 0.5% to 1% paraformaldehyde (optional)
  • 12 × 75–mm polystyrene tubes
  • Flow cytometer with either a single 488‐nm emission laser or both 488‐ and 635‐nm laser lines, four fluorescence detectors, and appropriate filter sets for detection of FITC, PE, and PerCP, PE‐Cy5, or PerCP‐Cy5.5, in addition to either APC or PE‐Texas Red
  • Software program for the analysis of flow cytometry FCS files (e.g., Paint‐A‐Gate Pro; Becton Dickinson)
  • Additional reagents and equipment for assessing absolute leukocyte counts (number of cells per microliter; appendix 3A)

Alternate Protocol 1: Flow Cytometric Enumeration of PB Dendritic Cells Using Microbeads as an Internal Reference to Derive Absolute Cell Counts

  • Flow‐Count beads (Beckmann Coulter) with fluorescence emission in green, orange, and red (i.e., 515 to 650 nm) once excited with a 488‐nm laser line
  • Additional reagents and equipment for reverse pipetting (unit 6.4)

Support Protocol 1: Immunophenotypic Characterization of PB Dendritic Cells by Flow Cytometry

  • Additional fluorochrome‐conjugated mAbs (unit 4.2):
  •  PE‐conjugated mAbs directed against the antigen under study
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Figures

Videos

Literature Cited

Literature Cited
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