Isolation of Dendritic Cells

Kayo Inaba1, William J. Swiggard2, Ralph M. Steinman2, Nikolaus Romani3, Gerold Schuler4, Carine Brinster5

1 Kyoto University, Kyoto, Japan, 2 The Rockefeller University, New York, New York, 3 University of Innsbruck, Innsbruck, Austria, 4 University of Erlangen, Erlangen, Germany, 5 IRCL (Institut de Recherche sur le Cancer de Lille), INSERM Unité 837, Lille, France
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 3.7
DOI:  10.1002/0471142735.im0307s86
Online Posting Date:  August, 2009
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Abstract

This unit presents two methods for preparing dendritic cells (DCs), a highly specialized type of antigen‐presenting cell (APC). The first method involves the isolation of DCs from mouse spleen, resulting in a cell population that is highly enriched in accessory cell and APC function. A support protocol for collagenase digestion of splenocyte suspensions is described to increase the yield of dendritic cells. The second method involves generating large numbers of DCs from mouse bone marrow progenitor cells. In that technique, bone marrow cells are cultured in the presence of granulocyte/macrophage colony‐stimulating factor (GM‐CSF) to yield 5–10 × 106 cells, 60% of which express DC surface markers (e.g., B‐7‐2/CD86). Additional techniques for isolating DCs from mouse spleens or other mouse tissues, as well as from human tissues, are also discussed. Curr. Protoc. Immunol. 86:3.7.1‐3.7.19. © 2009 by John Wiley & Sons, Inc.

Keywords: purity; maturation; co‐stimulation; antigen‐presenting cells

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Enrichment of Dendritic Cells by Plastic Adherence and EA Rosetting
  • Support Protocol 1: Preparation of Collagenase‐Digested Splenocyte Suspension
  • Alternate Protocol 1: Enrichment of Dendritic Cells by Magnetic Selection
  • Basic Protocol 2: Generation of Dendritic Cells from Proliferating Mouse Bone Marrow Progenitors
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Enrichment of Dendritic Cells by Plastic Adherence and EA Rosetting

  Materials
  • Collagenase‐digested splenocyte suspension (see protocol 2)
  • Dense bovine serum albumin (BSA) solution (see recipe)
  • RPMI 1640 medium (e.g., Life Technologies), 4° and 37°C
  • Complete RPMI‐5 medium (see recipe), 4° and 37°C
  • Antibody‐coated sheep erythrocytes (EA; see recipe)
  • Primary antibody for flow cytometry analysis (Table 3.7.2)
  • Secondary antibody for flow cytometry analysis (fluorescein‐conjugated mouse anti–rat IgG and IgM; e.g., Boehringer Mannheim)
  • Beckman GH‐3.7 and Sorvall HS‐4 rotors (or equivalents)
  • 15‐ and 50‐ml conical polypropylene tubes
  • Autoclaved 9‐in. (∼23‐cm) Pasteur pipets, plugged with cotton and unplugged
  • 60‐mm‐diameter tissue culture dishes (e.g., Falcon)
  • Additional reagents and equipment for counting viable cells ( appendix 3B) and for flow cytometry (units 5.3& 5.4)
    Table 3.7.2   MaterialsMonoclonal Antibodies Useful in Routine Flow Cytometry of Murine Splenic Dendritic Cells

    Specificity a Hybridoma Antibody type ATCC no. Reference
    Lymphoid DC 33D1 Rat IgG2b TIB 227 Nussenzweig et al. ( )
    Thy‐1.2 B5‐5 Rat IgG2b   — Nussenzweig and Steinman ( )
    CD32 (FcγRII) 2.4G2 Rat IgG2b HB 197 Unkeless ( )
    CD45RA (B220) RA3‐3A1/6.1 Rat IgG2a TIB 146 Coffman and Weissman ( )
    Macrophage SER‐4 Rat IgG2a   — Crocker and Gordon ( )
    Macrophage F4/80 Rat IgG2b HB 198 Austyn and Gordon ( )
    MHC‐I M1/42 Rat IgG2a TIB 126 Springer ( )
    MHC‐II (I‐A/E) M5/114 Rat IgG2b TIB 120 Bhattacharya et al. ( )
    CD11b (Mac‐1) M1/70 Rat IgG2b TIB 128 Springer ( )
    CD11c N418 Armenian hamster IgG HB 224 Metlay ( )
    CD80 (B7‐1) 16‐10A1 Armenian Hamster IgG2 HB 301 Razi‐Wolf ( )
    CD86 (B7‐2) GL1 Rat IgG2a HB 253 Hathcock ( )
    CD40 HM40‐3 Armenian hamster IgM   — Turner et al. ( )
    CD8alpha (Ly‐2) 53‐6.7 Rat IgG2a TIB 105 Ledbetter and Herzenberg ( )
    CD4 GK1.5 Rat IgG2b TIB 207 Dialynas ( )
    Interdig. Cells (DEC‐205) NLDC‐145 Rat IgG2a   — Kraal et al. ( )

     aAbbreviations: DC, dendritic cells; interdig., interdigitating.

Support Protocol 1: Preparation of Collagenase‐Digested Splenocyte Suspension

  Materials
  • 4000 U/ml collagenase D (see recipe), thawed and placed on ice
  • Hanks' balanced saline solution (HBSS), sterile, with Ca2+ and Mg2+ ( appendix 2A; may also be purchased from Life Technologies)
  • Mouse spleens (unit 1.9)
  • Hypodermic needles, 22‐G × 1½‐in. (Becton Dickinson)
  • 10‐ and 5‐ml disposable syringes (e.g., Becton Dickinson)
  • 100‐mm‐diameter petri dishes (e.g., Falcon)
  • Two serrated, semimicro dissecting forceps, autoclaved (e.g., Roboz)
  • Autoclaved stainless‐steel screen: cut 5 × 5–cm squares from 40‐mesh stainless steel wire cloth, fold cut edges underneath, then bend up all 4 sides to form a shallow rectangular bowl; wrap in foil before autoclaving

Alternate Protocol 1: Enrichment of Dendritic Cells by Magnetic Selection

  • Liberase blendzyme II (from Roche Applied Science or see recipe), aliquots thawed and placed on ice
  • Bovine pancreas DNase I, grade II (from Roche Applied Science or see recipe), aliquots thawed and placed on ice
  • Ammonium chloride solution (unit 2.5; may also be purchased from Invitrogen)
  • FBS/PBS buffer: phosphate‐buffered saline (PBS; appendix 2A) containing 2% (v/v) fetal bovine serum (may be purchased from Invitrogen)
  • Purified anti–mouse CD16/32 (Fcγ receptors) MAb (clone 2.4G2; e.g. Pharmingen) or purified mouse IgG (e.g. Jackson Immunoresearch laboratories)
  • Antibodies used for depletion: Phycoerythrin (PE)‐conjugated anti–mouse CD49b/Pan‐NK (clone DX5; e.g.; Pharmingen) and PE‐coupled anti–mouse CD3 (ɛ chain) (clone 145‐2C11, hybridoma ATCC #CRL‐1975)
  • Magnetic Cell Sorting (MACS) buffer (e.g., Miltenyi biotech or see recipe)
  • Anti‐PE and anti‐CD11c microbeads (e.g., Miltenyi biotech)
  • Cell separation columns for depletion or positive selection and separators or automated cell separator (autoMACS; e.g., Miltenyi Biotech)
  • Additional reagents and equipment for preparing the liberase blendzyme II‐digested splenocytes ( protocol 2) and counting viable cells ( appendix 3B)

Basic Protocol 2: Generation of Dendritic Cells from Proliferating Mouse Bone Marrow Progenitors

  Materials
  • Mice, 6 to 7 weeks old (preferably male)
  • 70% ethanol
  • RPMI 1640 medium (e.g., Life Technologies), ice‐cold and room temperature
  • Ammonium chloride solution (unit 2.5)
  • Antibodies (hybridoma supernatants) for lysing lymphocytes (optional)—e.g: hybridoma RA3‐3A1 (for MAb to B220; ATCC #TIB 146), GK1.5, (for MAb to CD4; ATCC #TIB 207), 3.155 (for MAb to CD8; ATCC #TIB 211), and M5/114 (for MAb to MHC II; ATCC #TIB 120)—also see unit 2.5
  • Rabbit complement (Pel‐Freez)
  • Complete RPMI‐5 medium (see recipe)
  • Mouse GM‐CSF (mGM‐CSF): either purified recombinant proteins or conditioned medium from cell lines transduced with the mouse GM‐CSF gene (gift from Dr. A. Lanzavecchia, Basel, Switzerland)
  • Antibodies for flow cytometric detection of DCs: e.g., hybridoma supernatant for MHC class II (ATCC #T1B120), hybridoma supernatant for B7‐2/CD86 (Pharmingen), and MAb NLDC‐145 for DEC‐205 (ATCC #HB 290)
  • Dissecting equipment
  • Sterile gauze pads
  • 100‐mm petri dishes (e.g., Falcon)
  • 3‐ml syringes with 25‐G, ⅝‐in. (1.58‐cm) needles
  • 9‐in. (∼23‐cm) Pasteur pipets, plugged with cotton and autoclaved
  • Nytex filters (Mesh 3‐40/26; Tetko)
  • 15‐ and 50‐ml conical polypropylene tubes
  • Beckman GH‐3.7 rotor (or equivalent)
  • 37°C water bath
  • 24‐well tissue culture plates (e.g., Corning)
  • Additional reagents and equipment for preparation of mouse bone marrow cells (unit 6.4 and protocol 2), counting viable cells ( appendix 3B), and flow cytometry (units 5.3& 5.4)
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Figures

Videos

Literature Cited

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Key References
   Agger et al., 1990. See above.
  Reviews the antigenic phenotype of mouse dendritic cells.
   Austyn, J.M. 1987. Lymphoid dendritic cells. Immunology 62:161‐170.
  General review of dendritic cell biology.
   Banchereau, J. and Steinman, R.M. 1998. Dendritic cells and the control of immunity. Nature. In press.
  Most recent review on the physiology of dendritic cells.
   Crowley et al., 1989. See above.
  Provides an extensive flow‐cytometric study of dendritic cells from spleen, thymus, and skin.
   Schuler, G. 1991. Epidermal Langerhans Cells. CRC Press, Boca Raton, Fla.
  Contains several articles on epidermal dendritic cells.
   Steinman, R.M. 1991. The dendritic cell system and its role in immunogenicity. Ann. Rev. Immunol. 9:271‐296.
  Review of the dendritic cell system in mouse and man.
   Steinman, R.M., Schuler, G., Romani, N., and Kaplan, G. 1987. Dendritic cells. In Atlas of Blood Cells: Function and Pathology (D. Zucker‐Franklin, M.F. Greaves, C.E. Grossi, and A.M. Marmont, eds.) pp. 359‐377. Lea & Febiger, Philadelphia.
  Cytologic study of dendritic cells in different organs and species.
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