Single-Cell Analysis: Methods and Protocols

249 Pages·2012·5.24 MB·English

by  Dino Di Carlo

M M B ™
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For further volumes:
http://www.springer.com/series/7651

Single-Cell Analysis
Methods and Protocols
Edited by
Sara Lindström
Department of Cell and Molecular Biology, Science For Life Laboratory,
Karolinska Institute, Stockholm, Sweden
Helene Andersson-Svahn
Division of Nanobiotechnology, AlbaNova University Centre,
Royal Institute of Technology, Stockholm, Sweden
Editors
Sara Lindström Helene Andersson-Svahn
Department of Cell and Molecular Biology Division of Nanobiotechnology
Science For Life Laboratory AlbaNova University Centre
Karolinska Institute Royal Institute of Technology
Stockholm, Sweden Stockholm, Sweden
[email protected]
ISSN 1064-3745 e-ISSN 1940-6029
ISBN 978-1-61779-566-4 e-ISBN 978-1-61779-567-1
DOI 10.1007/978-1-61779-567-1
Springer New York Heidelberg Dordrecht London
Library of Congress Control Number: 2012930263
© Springer Science+Business Media, LLC 2012
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Preface
Powerful tools for detailed cellular studies are emerging, increasing our knowledge of the
ultimate target of all drugs: the living cell. Today, cells are commonly analyzed en masse,
with thousands of cells per sample, yielding results on the average response of the cells.
However, cellular heterogeneity implies that in order to learn more about cellular behavior,
it is important to study how individual cells respond, one by one.
There are many conventional methods available for the study of single cells, such as
fl ow cytometry, microscopy, capillary electrophoresis, and laser capture microdissection.
These methods are typically robust, and their use has taught us almost everything we know
about single-cell behavior in different environments. Researchers within the fi eld of Life
Science are continuously developing exciting new methods for analyzing entire cells on the
proteomic, genomic, and transcriptomic level. Recently, miniaturized solutions have
emerged as exciting and complementary ways of analyzing individual cells. With these
microdevices and smaller volumes, experimental platforms are moving closer and closer to
the size and volume of a single cell. Miniaturization can, for example, enable: (1) limited
dilution of the cell content, (2) simultaneous analysis of several samples, and (3) an increase
in experimental control by compartmentalization of single cells in droplets or wells. The
methods described in this book include a few examples of conventional methods and several
examples of miniaturized methods.
This book aims to serve as an update on the fi eld of single-cell analysis, where the latest
fi ndings and applications are described in detail. The book is mainly not a review (although
some review chapters are included); instead, it offers detailed protocols on how to carry out
the methods in the laboratory. The invaluable “notes section” of each protocol chapter
includes the kinds of tips and tricks that are crucial for succeeding with the experiments and
yet are often left out of scientifi c publications. We believe that miniaturized systems are
highly suitable for analyzing individual cells, but we are also aware that they often demand
great laboratory skills and sometimes are far from “plug‘n’play” systems that can be easily
run by any end-user. By providing the readers with in-depth laboratory protocols, we hope
that these methods can be made much more accessible and user-friendly. The book contains
chapters written by leaders in the fi eld and provides a fi rm background for anyone working
with single-cell analysis. We hope that you will enjoy reading this book, which we believe is
a very timely volume in the M ethods in Molecular Biology series.
Below follows a short introduction to the book. The fi rst chapter gives an introduction
to single-cell analysis and discusses the huge progress and interest in the fi eld. The three
subsequent chapters deal with conventional techniques; Chap. 2 is a short review, introduc-
ing fl ow cytometry and microscopy as methods for analyzing single cells, Chap. 3 describes
how peptide products from single cells can be identifi ed using capillary electrophoresis and
liquid chromatography-mass spectrometry, and Chap. 4 explains how laser capture micro-
dissection can be used for gene expression analysis in single blastocysts.
v
vi Preface
The second part of the book deals with miniaturized solutions for single-cell analysis.
The fi fth chapter describes a method for culturing single cells into clones in order to study
their heterogeneity in terms of proliferation. Chapter 6 demonstrates a method for encod-
ing the cell’s electrical properties, and can be used to identify cells that are electrically dis-
tinct from a background population. The protocol in Chap. 7 describes how individual cells
are trapped in micrometer-size structures within a microchip, exposed in situ to a high
electric fi eld and loaded with plasmids. In Chap. 8, we learn more about how to resemble
cellular microenvironments by trapping and culturing cells in a microfl uidic channel with a
build-in pillar array. The ninth chapter describes how padlock probes and rolling circle
amplifi cation can be used to detect DNA sequences within comet preparations, and in
Chap. 10, we learn more about ways to analyze single cells using high-throughput droplet
technologies. Chapter 11 demonstrates a method for screening antigen-specifi c antibody
secreting cells using microwell-array chips. The twelfth chapter provides a generic protocol
for the analysis of fi xed and living single eukaryotic cells, including the considerations
required to build a Raman Tweezers system. Chapter 13 describes a method for single-cell
microinjection on nonadherent and adherent cells. In Chap. 14, we learn about how ultra-
sound can be combined with microfl uidics and microplates for particle and cell manipula-
tion approaching the single-cell level.
The third and fi nal part of the book contains two review chapters. In Chap. 15, we can
read about a rapidly growing fi eld and emerging promising technologies that now enable
sensitive “single-cell-omics” analysis. Lastly, Chap. 16 presents an engineering approach for
integrated single-cell analysis that uses interchangeable modular operations to provide a
comprehensive characterization of the phenotypic, functional, and genetic variations for
individual cells.
Finally, we wish to thank all the authors that have contributed to this book; it is thanks
to them that a volume on single-cell analysis could be realized. They have kindly shared
their expertise by describing the exciting methodologies that they are working on, giving
others a chance to fully understand and to try the methods themselves. In particular, we
wish to thank them for generously, including all their “in-house expertise” in the Notes
sections, containing the many tips and tricks that are needed in order to get the most out
of the described assays. To the readers, if you have questions or comments that are not
covered in the book, please do not hesitate to contact us for complementary information.
We hope that you will enjoy reading this book and fi nd it useful to learn more about the
methods described and the available assays for single-cell analysis. Our aim is to encourage
you to explore new ways of studying cells and help lead you to new discoveries.
Stockholm, Sweden Sara Lindström, Ph.D.
Helene Andersson-Svahn, Ph.D.
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Introduction: Why Analyze Single Cells?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Dino Di Carlo, Henry Tat Kwong Tse, and Daniel R. Gossett
PART I CONVENTIONAL METHODS
2 Flow Cytometry and Microscopy as Means of Studying
Single Cells: A Short Introductional Overview . . . . . . . . . . . . . . . . . . . . . . . . 13
Sara Lindström
3 Identification of Enzyme-Converted Peptide Products
from Single Cells Using Capillary Electrophoresis and Liquid
Chromatography-Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Robert B. Brown, Johannes A. Hewel, Andrew Emili,
and Julie Audet
4 Laser Capture Microdissection for Gene Expression Analysis
of Specific Cell Populations in Single Blastocysts. . . . . . . . . . . . . . . . . . . . . . . 29
Ward De Spiegelaere, Muriel Filliers, and Ann Van Soom
PART II MINIATURIZED SOLUTIONS
5 Single-Cell Culture in Microwells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Sara Lindström and Helene Andersson-Svahn
6 Isodielectric Separation and Analysis of Cells. . . . . . . . . . . . . . . . . . . . . . . . . . 53
Michael D. Vahey and Joel Voldman
7 Single Cell Electroporation Using Microfluidic Devices. . . . . . . . . . . . . . . . . . 65
Séverine Le Gac and Albert van den Berg
8 Perfusion Culture of Mammalian Cells in a Microfluidic Channel
with a Built-In Pillar Array . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Chi Zhang
9 Padlock Probes and Rolling Circle Amplification for Detection
of Repeats and Single-Copy Genes in the Single-Cell Comet Assay . . . . . . . . . 95
Sara Henriksson and Mats Nilsson
10 Droplet Microfluidics for Single-Cell Analysis . . . . . . . . . . . . . . . . . . . . . . . . . 105
Eric Brouzes
11 Screening of Antigen-Specific Antibody-Secreting Cells. . . . . . . . . . . . . . . . . . 141
Hiroyuki Kishi, Aishun Jin, Tatsuhiko Ozawa, Kazuto Tajiri,
Tsutomu Obata, and Atsushi Muraguchi
vii
viii Contents
12 Analysis of Single Eukaryotic Cells Using Raman Tweezers. . . . . . . . . . . . . . . 151
Elsa Correia Faria and Peter Gardner
13 Single-Cell Microinjection Technologies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Yan Zhang
14 Ultrasonic Manipulation of Single Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Martin Wiklund and Björn Önfelt
PART III REVIEWS ON CHOSEN SUBJECTS WITHIN THE FIELD
15 Expanding the Horizons for Single-Cell Applications
on Lab-on-a-Chip Devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Soo Hyeon Kim, Dominique Fourmy, and Teruo Fujii
16 Analytical Technologies for Integrated Single-Cell Analysis
of Human Immune Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
Ayça Yalçın, Yvonne J. Yamanaka, and J. Christopher Love
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
Contributors
HELENE ANDERSSON-SVAHN (cid:129) Division of Nanobiotechnology,
AlbaNova University Centre, Royal Institute of Technology , Stockholm , Sweden
JULIE AUDET (cid:129) Institute of Biomaterials and Biomedical Engineering (IBBME),
University of Toronto , Toronto , ON , Canada
ALBERT VAN DEN BERG (cid:129) BIOS, The Lab-on-a-Chip Group, MESA+ Institute
for Nanotechnology, University of Twente , Enschede , The Netherlands
ERIC BROUZES (cid:129) Department of Biomedical Engineering, Stony Brook University,
Stony Brook , NY , USA
ROBERT B. BROWN (cid:129) Institute of Biomaterials and Biomedical Engineering (IBBME),
University of Toronto , Toronto , ON , Canada
DINO DI CARLO (cid:129) Department of Bioengineering, University of California,
Los Angeles , CA , USA
ANDREW EMILI (cid:129) Banting and Best Department of Medical Research ,
University of Toronto , Toronto , ON , Canada
ELSA CORREIA FARIA (cid:129) Manchester Interdisciplinary Biocentre, School of Chemical
Engineering and Analytical Science, The University of Manchester, Manchester , UK
MURIEL FILLIERS (cid:129) Department of Reproduction, Obstetrics, and Herd Health,
Faculty of Veterinary Medicine, Ghent University, Merelbeke , Belgium
DOMINIQUE FOURMY (cid:129) LIMMS/CNRS-IIS , Institute of Industrial Science,
University of Tokyo , Tokyo , Japan
TERUO FUJII (cid:129) LIMMS/CNRS-IIS, JST-CREST , Institute of Industrial Science,
University of Tokyo , Tokyo , Japan
SÉVERINE LE GAC (cid:129) BIOS the Lab-on-a-Chip Group, MESA+ Institute
for Nanotechnology, University of Twente , Enschede , The Netherlands
PETER GARDNER (cid:129) Manchester Interdisciplinary Biocentre, School of Chemical
Engineering and Analytical Science, The University of Manchester, Manchester , UK
DANIEL R. GOSSETT (cid:129) Department of Bioengineering, University of California,
Los Angeles , CA , USA
SARA HENRIKSSON (cid:129) Department of Genetics and Pathology , Uppsala University,
Uppsala , Sweden
JOHANNES A. HEWEL (cid:129) Banting and Best Department of Medical Research ,
University of Toronto , Toronto , ON , Canada
AISHUN JIN (cid:129) Department of Immunology , College of Basic Medical Science,
Harbin Medical University , Nangang, Harbin , China
SOO HYEON KIM (cid:129) JST-CREST , Institute of Industrial Science, University of Tokyo,
Tokyo , Japan
HIROYUKI KISHI (cid:129) Department of Immunology , Graduate School of Medicine
and Pharmaceutical Sciences, University of Toyama , Toyama , Japan
ix