Differentiation of Peripheral Blood Monocytes into Dendritic Cells

David W. O'Neill1, Nina Bhardwaj1

1 New York University School of Medicine, New York, New York
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 22F.4
DOI:  10.1002/0471142735.im22f04s67
Online Posting Date:  July, 2005
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Abstract

Dendritic cells (DCs) are potent antigen‐presenting cells (APC) that are important in the initiation and control of cellular immune responses. Commonly used in T cell–stimulation experiments, DCs are typically “matured” in vitro with microbial products or proinflammatory cytokines, and then loaded with antigens from any number of sources, including peptides, whole proteins, cell lysates, RNA, microbes, or killed tumor cells. This unit presents a simple and commonly used method for the generation of mature human dendritic cells—differentiating them from peripheral blood monocytes.

Keywords: antigen presenting cells; dendritic; human; monocytes

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • Peripheral blood mononuclear cells (PBMC; unit 7.1)
  • RPMI‐10 (see recipe)
  • RPMI 1640 medium (e.g., Invitrogen; no serum or other additives), prewarmed to 37°C
  • PBS/5% albumin (see recipe)
  • 400 IU/µl IL‐4 (see recipe)
  • 100 IU/µl GM‐CSF (see recipe)
  • 100× monocyte conditioned medium (MCM) mimic (see recipe)
  • Heat‐inactivated human AB serum containing 10% (v/v) DMSO
  • Isopropanol
  • Liquid nitrogen
  • 10‐cm tissue culture dishes (BD Falcon)
  • Inverted microscope
  • 15‐ and 50‐ml conical polypropylene centrifuge tubes
  • Tabletop centrifuge
  • 6‐well tissue culture plates (BD Falcon)
  • 1.0 or 1.8‐ml cryovials
  • “Mr. Frosty” Cryo 1°C freezing container (Nalgene)
  • Liquid nitrogen freezer or equivalent
  • Additional reagents and equipment for determining cell viability by trypan blue exclusion ( appendix 3B)
NOTE: All solutions and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.
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Figures

Videos

Literature Cited

Literature Cited
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