Methods in
Molecular Biology 1423
Elodie Segura
Nobuyuki Onai Editors
Dendritic Cell
Protocols
Third Edition
,METHODS IN MOLECULAR BIOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For further volumes:
http://www.springer.com/series/7651
,
, Dendritic Cell Protocols
Third Edition
Edited by
Elodie Segura
Pavillon Pasteur, Institut Curie INSERM U932, Paris, France
Nobuyuki Onai
Department of Biodefense, Tokyo Medical and Dental University, Tokyo, Japan
,Editors
Elodie Segura Nobuyuki Onai
Pavillon Pasteur Department of Biodefense
Institut Curie INSERM U932 Tokyo Medical and Dental University
Paris, France Tokyo, Japan
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-3604-5 ISBN 978-1-4939-3606-9 (eBook)
DOI 10.1007/978-1-4939-3606-9
Library of Congress Control Number: 2016939414
© Springer Science+Business Media New York 2016
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,Preface
Dendritic cells are fascinating cells but can be challenging to work with. In this new edition
of Dendritic Cell Protocols, we aimed at complementing the previous edition in order to
provide both beginners and more experienced researchers a choice of methods to isolate
and analyze dendritic cells. An introductory review provides an overview of recent advances
in the characterization of DC subsets in mouse and human.
Dendritic cells are rare, fragile, and their isolation is often a tedious procedure. For
those who wish to generate dendritic cells in vitro, two chapters provide methods to culture
human and mouse dendritic cells. Several chapters are devoted to protocols for the isolation
of dendritic cells from various organs and tissues (lymphoid organs, intestine, skin, lung,
liver), both in mouse and human. In addition, a chapter describes the isolation of dendritic
cell progenitors from mouse, and another chapter the purification of dendritic cells from
human blood.
Dendritic cells are often described as conductors of the immune response and, as such,
perform a wide range of functions. We have compiled cutting-edge methods for the analysis
of dendritic cell properties ex vivo. Some of these methods can be applied to dendritic cells
from any species. We also included a “bioinformatics tutorial” chapter for the analysis of
dendritic cell transcriptome by nonexperts.
In vivo mouse studies have significantly contributed to our knowledge of dendritic cells
homeostasis, interactions with other immune cells, and division of labor between dendritic
cell subsets. The last section contains several protocols for the in vivo analysis of dendritic
cells through cell ablation, adoptive transfer, infection models, or in vivo imaging. Finally,
we provide a protocol for the generation of humanized mice for analyzing human dendritic
cells in a physiological setting.
The editors wish to thank all authors for their excellent contributions. We hope that
this Protocols book will be a valuable tool for a better understanding of dendritic cell
biology.
Paris, France Elodie Segura
Tokyo, Japan Nobuyuki Onai
v
,
,Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
PART I INTRODUCTION
1 Review of Mouse and Human Dendritic Cell Subsets . . . . . . . . . . . . . . . . . . . 3
Elodie Segura
PART II IN VITRO CULTURE OF DENDRITIC CELLS
2 In Vitro Generation of Human XCR1+ Dendritic Cells
from CD34+ Hematopoietic Progenitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Sreekumar Balan and Marc Dalod
3 Derivation and Utilization of Functional CD8+ Dendritic Cell Lines . . . . . . . . 39
Matteo Pigni, Devika Ashok, and Hans Acha-Orbea
PART III PURIFICATION AND ISOLATION OF DENDRITIC CELL SUBSETS
4 Isolation of Dendritic Cell Progenitor and Bone Marrow Progenitor
Cells from Mouse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Nobuyuki Onai and Toshiaki Ohteki
5 The Isolation and Enrichment of Large Numbers of Highly Purified
Mouse Spleen Dendritic Cell Populations and Their In Vitro Equivalents . . . . 61
David Vremec
6 Dendritic Cell Subset Purification from Human Tonsils and Lymph Nodes . . . 89
Mélanie Durand and Elodie Segura
7 Isolation and Identification of Conventional Dendritic Cell Subsets
from the Intestine of Mice and Men . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Charlotte L. Scott, Pamela B. Wright, Simon W.F. Milling,
and Allan McI Mowat
8 Isolation of Human Skin Dendritic Cell Subsets . . . . . . . . . . . . . . . . . . . . . . . 119
Merry Gunawan, Laura Jardine, and Muzlifah Haniffa
9 Isolation of Mouse Dendritic Cell Subsets and Macrophages from the Skin . . . 129
Camille Malosse and Sandrine Henri
10 Isolation of Conventional Dendritic Cells from Mouse Lungs . . . . . . . . . . . . . 139
Lianne van de Laar, Martin Guilliams, and Simon Tavernier
11 Purification of Human Dendritic Cell Subsets from Peripheral Blood . . . . . . . 153
Solana Alculumbre and Lucia Pattarini
vii
,viii Contents
12 Protocols for the Identification and Isolation of Antigen-Presenting
Cells in Human and Mouse Tissues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Naomi McGovern, Andreas Schlitzer, Baptiste Janela,
and Florent Ginhoux
PART IV EX VIVO FUNCTIONAL ANALYSIS OF DENDRITIC CELLS
13 Measurement of Export to the Cytosol in Dendritic Cells
Using a Cytofluorimetry-Based Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
Omar I. Vivar, Joao G. Magalhaes, and Sebastian Amigorena
14 Cross-Presentation Assay for Human Dendritic Cells. . . . . . . . . . . . . . . . . . . . 189
Elodie Segura
15 Analysis of Intracellular Trafficking of Dendritic Cell Receptors
for Antigen Targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Haiyin Liu, Claire Dumont, Angus P.R. Johnston, and Justine D. Mintern
16 Characterization of Dendritic Cell Subsets Through Gene
Expression Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
Thien-Phong Vu Manh and Marc Dalod
PART V IN VIVO ANALYSIS OF DENDRITIC CELLS
17 In Vivo Ablation of a Dendritic Cell Subset Expressing
the Chemokine Receptor XCR1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
Hiroaki Hemmi, Katsuaki Hoshino, and Tsuneyasu Kaisho
18 In Vivo Analysis of Intestinal Mononuclear Phagocytes . . . . . . . . . . . . . . . . . . 255
Caterina Curato, Biana Bernshtein, Tegest Aychek, and Steffen Jung
19 In Vivo Imaging of Cutaneous DCs in Mice . . . . . . . . . . . . . . . . . . . . . . . . . . 269
Gyohei Egawa and Kenji Kabashima
20 Analysis of Dendritic Cell Function Using Clec9A-DTR Transgenic Mice . . . . 275
Piotr Tetlak and Christiane Ruedl
21 Analysis of DC Functions Using CD205-DTR Knock-In Mice . . . . . . . . . . . . 291
Tomohiro Fukaya, Hideaki Takagi, Tomofumi Uto, Keiichi Arimura,
and Katsuaki Sato
22 Generation of Humanized Mice for Analysis of Human Dendritic Cells. . . . . . 309
Yasuyuki Saito, Jana M. Ellegast, and Markus G. Manz
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
, Contributors
HANS ACHA-ORBEA • Department of Biochemistry CIIL, University of Lausanne, Epalinges,
Switzerland
SOLANA ALCULUMBRE • INSERM U932 Immunity and Cancer, Paris, France; Institut
Curie, Centre de recherche, Paris, France
SEBASTIAN AMIGORENA • INSERM U932, Institut Curie, Centre de recherche, Paris, France
KEIICHI ARIMURA • Division of Immunology, Department of Infectious Diseases, Faculty
of Medicine, University of Miyazaki, Miyazaki, Japan
DEVIKA ASHOK • Department of Biochemistry CIIL, University of Lausanne, Epalinges,
Switzerland
TEGEST AYCHEK • Department of Immunology, The Weizmann Institute of Science, Rehovot,
Israel
SREEKUMAR BALAN • Centre d’Immunologie de Marseille-Luminy (CIML), Marseille, France;
INSERM U1104, Marseille, France; CNRS UMR7280, Marseille, France
BIANA BERNSHTEIN • Department of Immunology, The Weizmann Institute of Science,
Rehovot, Israel
CATERINA CURATO • Department of Immunology, The Weizmann Institute of Science,
Rehovot, Israel
MARC DALOD • Centre d’Immunologie de Marseille-Luminy (CIML)UNIV UM2,
Aix Marseille Université, Marseille, France; INSERM U1104, Marseille, France;
CNRS UMR7280, Marseille, France
CLAIRE DUMONT • Department of Biochemistry and Molecular Biology, Bio21 Molecular
Science and Biotechnology Institute, The University of Melbourne, Parkville, VIC,
Australia
MÉLANIE DURAND • INSERM U932, Paris, France; Institut Curie, Centre de recherche,
Paris, France
GYOHEI EGAWA • Department of Dermatology, Kyoto University Graduate School
of Medicine, Kyoto, Japan
JANA M. ELLEGAST • Division of Hematology, University Hospital Zurich, Zurich,
Switzerland
TOMOHIRO FUKAYA • Division of Immunology, Department of Infectious Diseases,
Faculty of Medicine, University of Miyazaki, Miyazaki, Japan
FLORENT GINHOUX • Singapore Immunology Network (SIgN), Agency for Science,
Technology and Research (A*STAR), Singapore, Singapore
MARTIN GUILLIAMS • Laboratory of Immunoregulation, VIB Inflammation Research
Center, Ghent University, Ghent, Belgium; Department of Biomedical Molecular, Biology,
Ghent University, Ghent, Belgium
MERRY GUNAWAN • Human DC Lab, Institute of Cellular Medicine, Newcastle University,
Newcastle upon Tyne, UK
MUZLIFAH HANIFFA • Human DC Lab, Institute of Cellular Medicine, Newcastle
University, Newcastle upon Tyne, UK
ix
