Isolation and Generation of Human Dendritic Cells

Smita Nair1, Gerald E. Archer1, Thomas F. Tedder1

1 Duke University Medical Center, Durham, North Carolina
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.32
DOI:  10.1002/0471142735.im0732s99
Online Posting Date:  November, 2012
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Abstract

Dendritic cells are highly specialized antigen‐presenting cells (APC), which may be isolated or generated from human blood mononuclear cells. Although mature blood dendritic cells normally represent ∼0.2% of human blood mononuclear cells, their frequency can be greatly increased using the cell enrichment methods described in this unit. More highly purified dendritic cell preparations can be obtained from these populations by sorting of fluorescence‐labeled cells. Alternatively, dendritic cells can be generated from monocytes by culture with the appropriate cytokines, as described here. In addition, a negative selection approach is provided that may be employed to generate cell preparations that have been depleted of dendritic cells to be used for comparison in functional studies. Curr. Protoc. Immunol. 99:7.32.1‐7.32.23. © 2012 by John Wiley & Sons, Inc.

Keywords: immunology; immunologic study in humans; in vitro assays for immune cell function; immune disease; innate immunity; antigen presentation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Dendritic Cells from Human Blood Mononuclear Cells
  • Alternate Protocol 1: Immunomagnetic Isolation of Dendritic Cells
  • Basic Protocol 2: Generation of Human Dendritic Cells from Monocytes
  • Alternate Protocol 2: Generation of Monocyte‐Derived Dendritic Cells from a Leukapheresis Pack
  • Basic Protocol 3: Depletion of Dendritic Cells by Negative Selection
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Dendritic Cells from Human Blood Mononuclear Cells

  Materials
  • Leukocyte‐enriched leukapheresis packs (leukopaks; i.e., 20 to 50 ml) or buffy coats, ≤24 hr old (from blood bank or North American Biologicals; see Critical Parameters)
  • Complete RPMI‐10 ( appendix 2A), room temperature and 37°C
  • RPMI 1640
  • 14.5% metrizamide solution (see recipe), room temperature
  • Dulbecco's PBS, Ca2+‐ and Mg2+‐free, 4°C
  • Primary antibody for flow cytometry analysis (see Table 7.32.1; optional)
  • Fluorescence‐labeled secondary antibody for flow cytometry analysis (optional)
  • 15‐ and 50‐ml conical polypropylene centrifuge tubes
  • 9‐in. (∼23‐cm) Pasteur pipets, with and without cotton plugs
  • 100‐mm tissue culture plates
  • Sorvall H1000B rotor (or equivalent)
  • Additional reagents and equipment for isolating mononuclear cells (unit 7.1), counting viable cells ( appendix 3B), rosetting with sheep red blood cells (unit 7.2), and flow cytometric analysis (units 5.3& 5.4) or assessment of functional activity by mixed leukocyte reaction (unit 7.10; both procedures optional)
    Table 7.2.1   MaterialsMonoclonal Antibodies Useful in Routine Flow Cytometric Analysis of Human Blood Dendritic Cells

    Antigen a Cell type where primarily expressed Reactivity with dendritic cells
    MHC class II Leukocytes +++++
    CD1c Thymocytes ++
    CD33 Monocytes ++
    CD40 B lymphocytes +
    CD83 Dendritic cells ++
    CD80 Antigen‐presenting cells ++
    CD86 Antigen‐presenting cells ++
    CD3 T lymphocytes None
    CD14 Monocytes None
    CD19 B lymphocytes None
    CD56 NK cells None

     aAll of the antigens listed are identified by MAbs that are widely available commercially. MAb reactivity is indicated on a relative linear scale, with +++++ indicating the highest levels of antigen expression.

Alternate Protocol 1: Immunomagnetic Isolation of Dendritic Cells

  • MAb mix including anti‐CD3, ‐CD14, ‐CD19, and ‐CD56 mAbs, each at 10× saturating concentrations (Table 7.32.1)
  • Goat anti–mouse IgG–coated magnetic beads (Dynabeads), washed (see unit 7.4)
  • Anti‐CD83 MAb at a 10× saturating concentration
  • Clinical rotator
  • Magnetic separation apparatus

Basic Protocol 2: Generation of Human Dendritic Cells from Monocytes

  Materials
  • Leukocyte‐enriched leukapheresis packs (leukopaks; i.e., 20 to 50 ml) or buffy coats, ≤24 hr old (from blood bank or North American Biologicals; see Critical Parameters)
  • Complete RPMI‐10 ( appendix 2A), 37°C
  • 0.2 mM EDTA in PBS, Ca2+ and Mg2+ free ( appendix 2A)
  • 14.5% metrizamide solution (see recipe), room temperature
  • Recombinant human granulocyte/macrophage colony stimulating factor (GM‐CSF; see recipe)
  • Recombinant human IL‐4 (see recipe)
  • Recombinant human TNF‐α (see recipe)
  • Inverted phase‐contrast microscope
  • Cotton‐plugged Pasteur pipet with a pipet bulb
  • Centrifuge
  • Sorvall H1000B rotor (or equivalent)
  • 15‐ml conical polypropylene centrifuge tube
  • Tissue culture flask
  • Additional reagents and equipment for isolation of peripheral blood monocytes (unit 7.1) followed by adherence to plastic (unit 7.6)

Alternate Protocol 2: Generation of Monocyte‐Derived Dendritic Cells from a Leukapheresis Pack

  Materials
  • Leukopak containing blood product
  • Alcohol swabs
  • Phosphate‐buffered saline (PBS; Invitrogen, cat. no. 14190‐144)
  • Ficoll‐Paque Premium (GE Healthcare, cat. no. 17‐5442‐02)
  • Trypan blue (Sigma, cat. no. T‐8154)
  • AIM V cell culture medium (Invitrogen, cat. no. 870112DK)
  • Recombinant human GM‐CSF [Berlex Laboratories, Leukine (sargramostim), recombinant granulocyte‐macrophage colony‐stimulating factor (GM‐CSF); see recipe]
  • Recombinant human IL‐4 (R&D Systems, cat. no. 204‐IL/CF; see recipe)
  • Ice
  • Cell stripper, enzyme‐free cell dissociation buffer (Cellgro, cat. no. 25‐056‐CI)
  • Recombinant human TNF‐α (R&D Systems, cat. no. 210‐TA/CF; see recipe)
  • Recombinant human IL‐1β (R&D Systems, cat. no. 201‐LB/CF; see recipe)
  • Recombinant human IL‐6 (R&D Systems, cat. no. 208‐IL/CF; see recipe)
  • Prostaglandin E 2 (PGE 2; see recipe)
  • Human AB serum (Valley Biomedical, cat. no. HP1022)
  • Cryoserv dimethyl sulfoxide (DMSO; Baxter, cat. no. 88‐850)
  • 50% Dextrose (Abbott Laboratories NDC 0074‐6648‐02), optional
  • Scalpel, sterile
  • 500‐ and 1000‐ml sterile Corning bottles
  • 50‐ml sterile polypropylene conical tubes
  • Sorvall RT‐6000D centrifuge (or equivalent)
  • Hemacytometer (Hausser, cat. no. 1492)
  • 75‐cm2 and 150‐cm2 cell culture flasks
  • Hemacytometer incubator maintained at 37°C and 5% CO 2
  • 2‐ml cryogenic vials (Corning, cat. no. 25704‐2)
  • Liquid nitrogen freezer
  • Additional reagents and equipment for isolating mononuclear cells (unit 7.1) and counting viable cells ( appendix 3B)

Basic Protocol 3: Depletion of Dendritic Cells by Negative Selection

  Materials
  • Complete RPMI‐10 ( appendix 2A)
  • CD83 MAb at a 10× saturating concentration
  • Goat anti–mouse IgG–coated magnetic beads (Dynabeads), washed (see unit 7.4)
  • Tissue culture flask or petri plate
  • 15‐ml conical polypropylene centrifuge tubes
  • Clinical rotator
  • Magnetic separation apparatus
  • Additional reagents and equipment for isolating mononuclear cells (unit 7.1) and counting viable cells ( appendix 3B)
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Figures

Videos

Literature Cited

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Key References
   Steinman, 1991. See above.
  Presents a general review of dendritic cell biology in mouse and man.
   Zhou and Tedder, 1995a. See above.
  Provides an extensive flow cytometric study of the antigenic phenotype of human dendritic cells.
   Zhou and Tedder, 1995b. See above.
  Surveys the cytokines and chemokines produced by human blood dendritic cells.
   Zhou and Tedder, 1996. See above.
  Details procedures for generating human dendritic cells from monocytes.
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