Methods in molecular biology vol 1591 t cell trafficking methods and protocols

Methods in
Molecular Biology 1591

George Edward Rainger
Helen M. Mcgettrick Editors

T-Cell
Trafficking
Methods and Protocols
Second 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:
/>

T-Cell Trafficking
Methods and Protocols
Second Edition

Edited by

George Edward Rainger
Institute of Cardiovascular Sciences, University of Birmingham, Birmingham, UK

Helen M. Mcgettrick
Institute of Inflammation and Ageing, College of Medicine and Dental Sciences,
University of Birmingham, Birmingham, UK


Editors
George Edward Rainger
Institute of Cardiovascular Sciences
University of Birmingham
Birmingham, UK

Helen M. Mcgettrick
Institute of Inflammation and Ageing
College of Medicine and Dental Sciences
University of Birmingham
Birmingham, UK

ISSN 1064-3745    ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-6929-6    ISBN 978-1-4939-6931-9 (eBook)
DOI 10.1007/978-1-4939-6931-9
Library of Congress Control Number: 2017932550
© Springer Science+Business Media LLC 2017
This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction

on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,
computer software, or by similar or dissimilar methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not
imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and
regulations and therefore free for general use.
The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to
be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty,
express or implied, with respect to the material contained herein or for any errors or omissions that may have been made.
The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Printed on acid-free paper
This Humana Press imprint is published by Springer Nature
The registered company is Springer Science+Business Media LLC
The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.


Preface
Welcome to the second edition of Methods and Protocols for assessing T cell Trafficking,
in the Methods in Molecular Biology series. The trafficking of T cells is relevant in numerous contexts. It occurs during population and maturation of T cells in the thymus and is
required for dissemination of antigen naïve T cells to the secondary lymphatic organs where
immune responses are initiated. Indeed, T cell trafficking within lymph nodes plays an
important role in the maturation of primary and secondary immune responses. Antigen
experienced effector T cells can undertake compartmentalized recirculation during immune
surveillance. In addition they are recruited to tissues during inflammation where they play
important roles in the inflammatory response. Importantly, we now recognize that inappropriate or persistent trafficking of T cells into such sites makes a major contribution to
the pathogenesis of immune-mediated inflammatory diseases which have an autoimmune
or chronic inflammatory component.
Thus the trafficking of T cells has both physiological and pathological relevance and
provides some challenging environments in which to make quantitative measurements.
This has seen the development of expertise which goes well beyond the standard laboratory
methodologies which can be supported by commercially available kits and reagents. The

methods in this edition have been developed by experts in T cell trafficking, who have spent
many years perfecting them. Each chapter contains a step-by-step guide to conducting the
assays, with useful hints to avoid common pitfalls. The volume is organized into three sections. The first addresses homeostatic T cell trafficking during thymic maturation, followed
by the subsequent colonization of and egress from secondary lymphoid organs. The second
addresses T cell trafficking during “normal” inflammatory and immune responses. Lastly,
we include a section on T cell trafficking in disease. Each section is headed by an informative and accessible introduction written by experts who are actively investigating the regulation of T cell trafficking in these different scenarios. We believe this will ensure that this
book will become an essential point of reference for those new to the field of T cell trafficking, or to those looking to expand their technical capabilities.
We would like to thank all the authors for their invaluable contributions and willingness
to share their expertise. Thanks also to Professor John Walker, the series editor, for guidance in the process of compiling the book.
G. Ed. Rainger is generously supported by the British Heart Foundation of the UK.
Helen M. Mcgettrick is supported by generous funds from Arthritis Research UK.
Birmingham, UK


George Edward Rainger
Helen M. Mcgettrick

v


Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
  1 Introduction to Homeostatic Migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mark C. Coles
  2 Analysis of Thymocyte Migration, Cellular Interactions, and Activation
by Multiphoton Fluorescence Microscopy of Live Thymic Slices . . . . . . . . . . . .
Jessica N. Lancaster and Lauren I.R. Ehrlich
  3 Visualizing and Tracking T Cell Motility In Vivo . . . . . . . . . . . . . . . . . . . . . . .

Robert A. Benson, James M. Brewer, and Paul Garside
  4 Graph Theory-Based Analysis of the Lymph Node Fibroblastic Reticular
Cell Network . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mario Novkovic, Lucas Onder, Gennady Bocharov,
and Burkhard Ludewig
  5 Visualizing Endogenous Effector T Cell Egress from the Lymph Nodes . . . . . .
Manisha Menon, Alexandre P. Benechet, and Kamal M. Khanna
  6 Introduction: T Cell Trafficking in Inflammation and Immunity . . . . . . . . . . .
Myriam Chimen, Bonita H.R. Apta, and Helen M. Mcgettrick
  7 Leukocyte Adhesion Under Hemodynamic Flow Conditions . . . . . . . . . . . . . .
Charlotte Lawson, Marlene Rose, and Sabine Wolf
  8 Endocrine Regulation of Lymphocyte Trafficking In Vitro . . . . . . . . . . . . . . . .
Bonita H.R. Apta, Myriam Chimen, and Helen M. Mcgettrick
  9 Mesenchymal Stromal Cells as Active Regulators of Lymphocyte
Recruitment to Blood Vascular Endothelial Cells . . . . . . . . . . . . . . . . . . . . . . .
Helen M. Mcgettrick, Lewis S.C. Ward, George Edward Rainger,
and Gerard B. Nash
10 Monitoring RhoGTPase Activity in Leukocytes Using Classic
“Pull-Down” Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Marouan Zarrouk, David Killock, Izajur Rahman, Jessica Davies,
and Aleksandar Ivetić
11 Utilizing Lentiviral Gene Transfer in Primary Endothelial Cells
to Assess Lymphocyte-Endothelial Interactions . . . . . . . . . . . . . . . . . . . . . . . .
Jasmeet S. Reyat, Michael G. Tomlinson, and Peter J. Noy
12 Introduction to Lymphocyte Trafficking in Disease . . . . . . . . . . . . . . . . . . . . .
Patricia F. Lalor and Elizabeth A. Hepburn
13 Using Ex Vivo Liver Organ Cultures to Measure Lymphocyte Trafficking . . . .
Benjamin G. Wiggins, Zania Stamataki, and Patricia F. Lalor

vii

1

9
27

43

59
73
85
101

121

143

155
169
177


viii

Contents

14 In Vitro and Ex Vivo Models to Study T Cell Migration Through
the Human Liver Parenchyma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Benjamin G. Wiggins, Konstantinos Aliazis, Scott P. Davies,
Gideon Hirschfield, Patricia F. Lalor, Gary Reynolds, and Zania Stamataki

15 Monitoring Migration of Activated T Cells to Antigen-Rich
Non-lymphoid Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Eleanor Jayne Ward, Hongmei Fu, and Federica Marelli-Berg
16 Tissue Digestion for Stromal Cell and Leukocyte Isolation . . . . . . . . . . . . . . . .
Saba Nayar, Joana Campos, Nathalie Steinthal, and Francesca Barone
17 T Cell Response in the Lung Following Influenza Virus Infection . . . . . . . . . .
Robert A. Benson, Jennifer C. Lawton, and Megan K.L. MacLeod

195

215
225
235

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249


Contributors
Konstantinos Aliazis  •  Centre for Liver Research, Institute for Immunology and
Immunotherapy, University of Birmingham, Birmingham, UK
Bonita H.R. Apta  •  Institute of Cardiovascular Sciences, College of Medicine and Dental
Sciences, University of Birmingham, Birmingham, UK
Francesca Barone  •  Centre for Translational Inflammation Research, Institute of
Inflammation and Ageing, College of Medical & Dental Sciences, University of
Birmingham Research Laboratories, Queen Elizabeth Hospital, Birmingham, UK
Alexandre P. Benechet  •  Division of Immunology, Transplantation and Infectious
Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy
Robert A. Benson  •  Centre for Immunobiology, Institute of Infection, Immunity and
Inflammation, The University of Glasgow, Glasgow, UK
Federica Marelli-Berg  •  William Harvey Research Institute—Heart Centre,

Barts and the London School of Medicine and Dentistry, Queen Mary University
of London, London, UK
Gennady Bocharov  •  Institute of Numerical Mathematics, Russian Academy of Sciences,
Moscow, Russian Federation
James M. Brewer  •  Institute of Infection, Immunity and Inflammation, College of
Medical, Veterinary and Life Sciences, Glasgow Biomedical Research Centre,
University of Glasgow, Glasgow, UK
Joana Campos  •  Centre for Translational Inflammation Research, Institute of
Inflammation and Ageing, College of Medical & Dental Sciences, University of
Birmingham Research Laboratories, Queen Elizabeth Hospital, Birmingham, UK
Myriam Chimen  •  Institute of Cardiovascular Sciences, College of Medicine and Dental
Sciences, University of Birmingham, Birmingham, UK
Mark C. Coles  •  Department of Biology, Centre for Immunology and Infection, University
of York, North Yorkshire, UK
Scott P. Davies  •  Centre for Liver Research, Institute for Immunology and
Immunotherapy, University of Birmingham, Birmingham, UK
Jessica Davies  •  Cytoskeleton/Membrane Signalling Research Group, Cardiovascular
Division, King’s College London, London, UK
Lauren I.R. Ehrlich  •  Department of Molecular Biosciences, Institute for Cellular
and Molecular Biology, The University of Texas at Austin, Austin, TX, USA
Hongmei Fu  •  William Harvey Research Institute—Heart Centre, Barts and the London
School of Medicine and Dentistry, Queen Mary University of London, London, UK
Paul Garside  •  Institute of Infection, Immunity and Inflammation, College of Medical,
Veterinary and Life Sciences, Glasgow Biomedical Research Centre, Wellcome Trust
Centre for Molecular Parasitology, Glasgow, UK
Elizabeth A. Hepburn  •  Department of Cellular Pathology, Cheltenham General
Hospital, Cheltenham, UK
Gideon Hirschfield  •  Centre for Liver Research, Institute for Immunology and
Immunotherapy, University of Birmingham, Birmingham, UK

ix


x

Contributors

Aleksandar Ivetić  •  Cytoskeleton/Membrane Signalling Research Group, Cardiovascular
Division, King’s College London, London, UK
Kamal M. Khanna  •  Department of Immunology, University of Connecticut Health,
Farmington, CT, USA
David Killock  •  Cytoskeleton/Membrane Signalling Research Group, Cardiovascular
Division, King’s College London, London, UK
Patricia F. Lalor  •  Centre for Liver Research, Immunity and Immunotherapy, Institute
of Biomedical Research, University of Birmingham, Birmingham, UK
Jessica N. Lancaster  •  Department of Molecular Biosciences, Institute for Cellular
and Molecular Biology, The University of Texas at Austin, Austin, TX, USA
Charlotte Lawson  •  Comparative Biomedical Sciences, Royal Veterinary College,
London, UK
Jennifer C. Lawton  •  Centre for Immunobiology, Institute of Infection, Immunity
and Inflammation, The University of Glasgow, Glasgow, UK
Burkhard Ludewig  •  Institute of Immunobiology, Kantonsspital St. Gallen, St. Gallen,
Switzerland
Megan K.L. MacLeod  •  Centre for Immunobiology, Institute of Infection, Immunity
and Inflammation, The University of Glasgow, Glasgow, UK
Helen M. Mcgettrick  •  Institute of Inflammation and Ageing, College of Medicine
and Dental Sciences, University of Birmingham, Birmingham, UK
Manisha Menon  •  Department of Immunology , University of Connecticut Health,
Farmington, CT, USA
Gerard B. Nash  •  Institute of Cardiovascular Sciences, University of Birmingham,

Birmingham, UK
Saba Nayar  •  Centre for Translational Inflammation Research, Institute of Inflammation
and Ageing, College of Medical & Dental Sciences, University of Birmingham Research
Laboratories, Queen Elizabeth Hospital, Birmingham, UK
Mario Novkovic  •  Institute of Immunobiology, Kantonsspital St. Gallen, St. Gallen,
Switzerland
Peter J. Noy  •  School of Biosciences, College of Life and Environmental Sciences, University
of Birmingham, Birmingham, UK
Lucas Onder  •  Institute of Immunobiology, Kantonsspital St. Gallen, St. Gallen,
Switzerland
Izajur Rahman  •  Cytoskeleton/Membrane Signalling Research Group, Cardiovascular
Division, King’s College London, London, UK
George Edward Rainger  •  Institute of Cardiovascular Sciences, University of
Birmingham, Birmingham, UK
Jasmeet S. Reyat  •  School of Biosciences, College of Life and Environmental Sciences,
University of Birmingham, Birmingham, UK
Gary Reynolds  •  Centre for Liver Research, Institute for Immunology and
Immunotherapy, University of Birmingham, Birmingham, UK
Marlene Rose  •  Harefield Hospital, Imperial College, London, UK
Zania Stamataki  •  Centre for Liver Research, Immunity and Immunotherapy, Institute
of Biomedical Research, University of Birmingham, Birmingham, UK
Nathalie Steinthal  •  Centre for Translational Inflammation Research, Institute of
Inflammation and Ageing, College of Medical & Dental Sciences, University of
Birmingham Research Laboratories, Queen Elizabeth Hospital, Birmingham, UK


Contributors

xi

Michael G. Tomlinson  •  School of Biosciences, College of Life and Environmental Sciences,
University of Birmingham, Birmingham, UK
Eleanor Jayne Ward  •  William Harvey Research Institute—Heart Centre, Barts and the
London School of Medicine and Dentistry, Queen Mary University of London, London, UK
Lewis S.C. Ward  •  Institute of Inflammation and Ageing, University of Birmingham,
Birmingham, UK
Benjamin G. Wiggins  •  Centre for Liver Research, Immunity and Immunotherapy,
Institute of Biomedical Research, University of Birmingham, Birmingham, UK
Sabine Wolf  •  Comparative Biomedical Sciences, Royal Veterinary College, London, UK
Marouan Zarrouk  •  Cytoskeleton/Membrane Signalling Research Group, Cardiovascular
Division, King’s College London, London, UK


Chapter 1
Introduction to Homeostatic Migration
Mark C. Coles
Abstract
Immune cell development and function occur in specialized immunological tissues, the function of which
requires active cell migration and interactions between hematopoietic cells and underlying networks of
stromal cells. These cells provide a scaffold on which immune cell migrate, provide microenvironments for
efficient antigen presentation, and provide signals required for immune cell recruitment and survival.
Technical advances in imaging technologies including multiphoton microscopy and 3D tissue reconstructions are being combined with computational approaches to provide new insights into the process of cell
migration and function in immunological tissues.
Key words Multiphoton, Modeling, 3D imaging, Migration, Thymus

1  Introduction
Order from Chaos: the key role of migration in immune system development, homeostasis, and function: Cellular migration is the driving
force behind mammalian immune system development and function. In humans this involves the continuous stochastic migration
of billions of cells within lymphoid tissues and between the 600
and 800 different peripheral lymph nodes. It is this rapid migration

of lymphocytes in and between tissues, and the subsequent interactions between innate and adaptive immune cells that determine
immune efficacy. Order, “immunity,” emerges from the chaotic
movement of immune cells, a process that has evolved over the last
500 million years to provide a system that permits efficient adaptive immune responses to unknown pathogens, providing the
capacity of very small numbers of lymphocytes to efficiently
respond in localized lymphoid tissues and develop long-term
memory to immunological challenges.
Over the last 120 years, immunologists have come to appreciate the role migration has in lymphoid tissue formation, immune
cell development, and function using a range of technologies from
light microscopy to radiolabeled cellular transfers in sheep and pigs

George Edward Rainger and Helen M. Mcgettrick (eds.), T-Cell Trafficking: Methods and Protocols, Methods in Molecular Biology,
vol. 1591, DOI 10.1007/978-1-4939-6931-9_1, © Springer Science+Business Media LLC 2017

1


2

Mark C. Coles

to lineage-specific fluorescent protein transgenic and knock-in
mice permitting 3-dimensional (3D) imaging of immune homeostasis and function. An appreciation of lymphocyte migration was
first visualized in 1896 by Saxer who described “wandering lymphocytes” in emergent lymph nodes, a cell type later identified
exactly 100 years later to be lymphoid tissue inducer cells [1].
Later work by Alexandre Maximow identified three different populations in lymph nodes, “wandering lymphocytes,” “resting wandering cells” (macrophages), and collagen-producing stromal
fibroblasts [2]. Classical work from the 1970s onwards defined
central principles of immune cell migration through blood vessels
into both lymphoid tissues through specialized vessels, high endothelial venules (HEV), lymphocyte entry into peripheral tissues,
and migration of immune cells from peripheral tissues to lymph

nodes and subsequent entry into circulation through draining lymphatic vessels [3]. Although these seminal experiments provided
key insights into migration between tissues, the role of migration
in tissues was not well understood. The emergence of multiphoton
imaging in the early 2000s provided a new technological platform
to provide insights into the scale of lymphocyte migration and the
kinetics of immune cell–APC interactions [4, 5]. Lymphocytes
were found to very rapidly migrate within tissues and interact with
antigen-presenting cells (T cell—dendritic cell, B cell -T cell, B
cell—follicular dendritic cells (FDC)) in lymphoid tissues responding to localized cues in their microenvironment produced by a 3D
meshwork of specialized stromal cells [6]. Fibroblastic reticular
cells (FRC), marginal reticular cells (MRC), and B cell zone stroma
including FDCs are specialized stromal sets that support lymphocyte homeostasis through the production of survival factors interleukin-­7 (IL-7) for T cells and BAFF for B cells; homeostatic
chemokines CCL19, CCL21 (FRC, MRC), and CXCL13 (MRC,
FDC); and signaling lipids (e.g., 7a,25 OH cholesterol) that control the positioning and migration of lymphocytes [7]. This process permits very rare antigen-specific cells as low as one in million
to effectively respond to antigen in the highly organized stromal
lymphoid tissue microenvironment.
Despite the plethora of data from imaging and omics technologies, many questions remain to be addressed on how migration is
regulated in lymphoid tissues through all stages of their development and function. Understanding the molecular, biophysical, and
cellular processes of immune cell migration is of clinical significance; targeting lymphocyte entry into (natalizumab: anti-VLA4)
and exit (fingolimod: S1PR antagonism) from tissues has shown
efficacy for the treatment of multiple sclerosis [8]. Adjuvants in
vaccines in part work by stimulating tissue remodeling in B cell follicles leading to germinal center reactions, an emergent behavior
driven by active cross talk between innate immune cells, activated


Introduction to Homeostatic Migration

3

lymphocytes, and stroma. Thus the development of new therapeutics and vaccines that can enhance protective immune responses to

pathogens, inhibit immune-mediated inflammatory disease pathology, and potentiate antitumor immune responses requires new
insights into mechanisms regulating immune cell migration including entry and exit from tissues and migration within those tissues.
Increasingly it has become clear that modifying the kinetics of
immune cell migration in lymphoid tissues is likely to be a potent
method to selectively target immune function with less off-target
effects. Through the development of novel biologics, linked nucleic
acids, small molecules, and adjuvants targeting receptors and signaling pathways that regulate immune cell movement it is possible
to specifically target mechanisms of immune cell migration. The
scientific and clinical need to understand cellular migration has led
to the development of new methodologies described in subsequent
chapters describing methodologies to image and quantify immune
cell migration and stromal networks that support their migration.

2  Migration in Immune Cell Development
The development of lymphocytes and immune tissues is a highly
dynamic process involving migration between tissues (e.g., bone
marrow to thymus) and the active movement and interactions of
cells within primary and secondary lymphoid tissues where they
undergo their development and maturation. Lymphocytes arise
from committed progenitors in the bone marrow, and B cells
undergo their development and maturation in bone marrow
migrating between specialized stromal niches that express IL-7
required for their survival and expansion and specialized galectin-1
expressing stromal niches that select for successful BCR rearrangements [9]. B cells then migrate to the spleen where they undergo
further maturation into mature naïve B cells. In contrast T cells
undergo development in the thymus, a specialized organ that permits selection of restricted T cell repertoire with low affinity to
self-MHC-peptide complex. This process involves the active migration and highly dynamic interactions of developing thymocytes in
key anatomical niches in the thymus where they undergo a series of
steps and check points in their development including pre-TCR
selection of CD4−CD8− thymocytes in the paracortex, positive

selection of CD4+CD8+ cells on cortical epithelium, and the negative selection of single-positive thymocytes by medullary microenvironment [10]. This process is guided in part by localized
production of chemokines by stromal and epithelial cells and interactions with specialized dendritic cells and macrophages within the
thymus microenvironment [11].


4

Mark C. Coles

Migration not only dictates the development of immune cells;
it has an essential role in the formation of primary and secondary
lymphoid tissues. The development of the thymus involves not
only infiltration of the developing thymus by lymphocyte progenitors but also the active migration of epithelium and neural crest-­
derived stroma [12]. Likewise, the development of lymph node
and Peyer’s patch anlagen are active processes involving infiltration
by specialized lymphoid tissue initiator and inducer cells; their
active migration and interactions with localized mesenchyme initiate the process of lymphoid stromal cell maturation into stromal
subsets found in adult lymphoid tissues. This process is regulated
by the same adhesion molecules (VCAM, ICAM) and chemokines
(CXCL13, CCL19/21) that maintain the function of adult lymphoid tissues [13].

3  Migration in Immune Cell Function: Entry to Exit
Under homeostatic conditions lymphocytes continually recirculate, entering lymph nodes through HEVs and exiting through
efferent lymphatics, with a transit time of 10–22 hrs dependent on
cell type [14]. Activated dendritic cells (DC) and antigen enter
through the afferent lymphatics, with antigen either entering
through conduit network (<70 kD) or actively transported by subcapsular macrophages. The migration of DCs is a CCR7-dependent
process requiring formation of an active chemotactic gradient of
CCL21 generated by expression of the atypical chemokine receptor CCRL1 (ACKR4) on lymphatic endothelial cells [15]. Likewise,
the entry and migration of T cells in the LN cortex are dependent

on localized expression of CCL19/21 by fibroblastic reticular cells
and endothelial cells; in contrast B cells respond to CXCL13
expression by B cell stroma. Expression of stromal chemokines
leads to the phenomena of chemokinesis, where the migration rate
of lymphocytes is increased when in the presence of chemokine
receptor signaling [16]. This process has a key role in regulating
the efficacy of immune responses as it is the chance encounter of
lymphocyte with antigen-expressing DC that drives the efficacy of
T cell responses and capacity of B cells to encounter antigen-complement complexes on FDCs.
The short transit times of naïve lymphocytes in LNs require
active mechanisms controlling their egress, in contrast to naïve
cells activated lymphocytes that are retained during the early stages
of an immune response in a CD69-dependent process. This egress
of lymphocytes is dependent on sphingosine-1-phosphate (S1P)
production, a small signaling lipid that activates S1P receptors
expressed by lymphocytes. Modulation of S1P1 (S1P receptor 1)
expression through the synthetic pro-drug FTY720 that binds to


Introduction to Homeostatic Migration

5

several S1P receptors drives receptor internalization and destruction, blocks lymphocyte egress from LN and leads to lymphopenia
in the blood [17]. Similar to CCL19–ACKR4-mediated chemotactic gradient generation, localized S1P internalization and
destruction lead to chemotactic gradient formation. The expression of the S1P transporter ABCC7 and S1P lyase SGPL1 by
MRCs leads to the destruction of S1P in the marginal zone surrounding the LN lymphatics providing for the generation of very
tight S1P gradients permitting controlled lymphocyte egress [18].

4  Imaging Immune Responses: Multiphoton Microscopy

Although immune cells were known to migrate within tissues the
dynamics of this process was not fully appreciated until the development of multiphoton microscopy (MP). This technology revolutionized immunology by permitting imaging of immune cells
deep within tissues. This was possible due to some unique features
including deep tissue penetration of near-infrared light, lowered
phototoxicity, and generation of second harmonics that permit
visualization of the secondary structure of collagen. Utilizing both
tissue explants and in vivo imaging of lymph nodes the migration
and behaviors of lymphocytes have been quantified under homeostatic conditions, antigenic stimulation, inflammation, pathogen
infection using fluorescent dyes, and lineage-specific fluorescent
protein mice in combination with mice where key pathways are
inhibited using either tissue-specific gene knockout mice, inhibitory antibodies, or small-molecule inhibitors [19]. Using these
approaches, the relative role of GPCR signaling pathways (chemokine receptors, lipid-signaling molecules (EBI2, S1PR)), signaling
pathways regulating lymphocyte migration in lymph nodes, and
adhesion molecules in cell migration has been extensively studied
(see Chapters 3, 5, and 17). This technology has been applied by
Lauren Ehrlich (see Chapter 2) and Ellen Robey to provide new
insights into the process of T cell migration in the thymus and
interactions that drive positive and negative selection in the thymus
[20, 21].

5  3-Dimensional Tissue Imaging
Immune homeostasis and antigen driven initiation of adaptive
immune responses occur within the 3D structure of lymphoid
tissues. Although multiphoton imaging is a powerful technique
to study active immune cell migration it is inherently limited in
the depth penetration of light into dense tissues like lymph nodes
and the capacity to analyze more than four fluorescent signals is


6

Mark C. Coles

limited due to the design of most commercial microscope systems. A number of techniques when combined with optical clearing technologies including optical projection tomography and
light sheet microscopy have revolutionized the ability to quantify
large volumes of tissues in 3D using multiple different antibodies
[22]. When applied to LNs it has provided 3D reconstructions of
stromal topology and insights into pathogen-mediated LN
remodeling [23].

6  Quantifying Biology and the Emergence of Modeling Technologies
Through the quantification of multiphoton images, it is possible to
obtain a large amount of quantitative information on immune cell
migration and interactions including the velocity, meandering
index, turning angles, duration and timing of interactions, and
physiological outcomes of these interactions. This has provided the
data for modeling immune responses; this approach has provided
new insights into the data and provided models for how immune
cells migrate and interact [24]. T cells migrate in the context of
stromal networks. Analysis of simultaneous multiphoton imaging
of stromal networks and lymphocytes provided evidence for a
direct role of stromal cells in lymphocyte migration in LNs [6].
This has been extended through the very elegant work by Burkhard
Ludewig (see Chapter 4) and colleagues through generation of a
methodology to quantify 3D stromal networks in LNs using graph
theory [25]. Together these data provide the basis for using model-­
driven experimentation as a methodology to understand mechanisms of immune function [26]. This has been used to great effect
to understand how lymph nodes remodel during LCMV infection
[27] and mechanisms driving the regulation of the germinal center
response [28, 29]. This approach has strong potential to provide

new insights into mechanisms of immune cell migration and targeting these processes to develop the next generations of
immunotherapeutics.

7  Conclusions
The development of 3D imaging when combined with a quantitative approach is providing new insights into mechanisms regulating immune cell migration. The chapters that follow detail
methodologies to image mouse lymph nodes and thymus in 3D
and quantify the immune responses. The application of whole-­
tissue clearing and imaging in these protocols provides the capacity
to generate 3D maps of immune cells within lymph nodes, and
when combined with powerful mathematical techniques provides a
platform to produce new insights into immune tissue function.


Introduction to Homeostatic Migration

7

References
1.Saxer F (1896) Uber die Entwickelung und
den Bau der normalen Lymphdrusen und die
Entstehung der roten und weissen blutkorperchen. Anta Hefte 6:347–532
2. Maximow A (1924) Relations of blood cells to
connective tissues and endothelium. Physiol
Rev 4:533–563
3.
Ager A, Coles MC, Stein JV (2011)
Development of lymph node circulation and
homing mechanisms. In: Balogh P (ed)
Development biology of peripheral lymphoid
organs. Springer, Berlin, pp 75–94

4.
Germain RN, Miller MJ, Dustin M,
Nussenzweig MC (2006) Dynamic imaging of
the immune system: progress, pitfalls and
promise. Nat Rev Immunol 6:497–507
5.Mempel TR, Scimone ML, Mora JR, von
Adrian UH (2004) In vivo imaging of leukocyte trafficking in blood vessels and tissues.
Curr Opin Immunol 16(4):406–417
6.Bajenoff M, Egen JG, Koo LY, Laugier JP,
Brau F, Glaichenhaus N, Germain RN (2006)
Stromal cell networks regulate lymphocyte
entry, migration, and territoriality in lymph
nodes. Immunity 25(6):989–1001
7.Junt T, Scandella E, Ludewig B (2008) Form
follows function: lymphoid tissue microarchitecture in antimicrobial immune defence. Nat
Rev Immunol 8:764–775
8.Dendrou CA, Fugger L, Friese MA (2015)
Immunopathology of multiple sclerosis. Nat
Rev Immunol 15:545–558
9.Mourcin F, Breton C, Tellier J, Narang P,
Chasson L, Jorquera A, Coles M, Schiff C,
Mancini SJ (2011) Galectin-1-expressing stromal cells constitute a specific niche for pre-BII
cell development in the mouse bone marrow.
Blood 117(24):6552–6561
10. Spits H (2002) Development of αβ T cells in the
human thymus. Nat Rev Immunol 2:760–772
11.Hu Z, Lancaster JN, Ehrlich LI (2015) The
contribution of chemokines and migration to
the induction of central tolerance in the thymus. Front Immunol 6:398

12.Foster KE, Gordon J, Cardenas K, Veiga-­
Fernandes H, Makinen T, Grigorieva E,
Wilkinson DG, Blackburn CC, Richie E,
Manley NR, Adams RH, Kioussis D, Coles MC
(2010) EphB-ephrin-b2 interactions are
required for thymus migration during organogenesis. Proc Natl Acad Sci U S A
107(30):13414–13419

13.Veiga-Fernendes H, Coles MC, Foster KE,
Patel A, Williams A, Natarajan D, Barlow A,
Pachnis V, Kioussis D (2007) Tyrosine kinase
receptor RET is a key regulator of Peyer’s patch
organogenesis. Nature 446(7135):547–551

14.Mandi, JN, Liou, R, Klauschen, F, Vrisekoop,
N, Monteiro, JP, Yates, AJ, Haung, AY,
Germain, R (2012) Quantification of lymph
node transit times reveals differences in antigen
surveillance strategies of naive CD4+ and
CD8+ T cells. Proc Natl. Acad Sci USA
109(44):18036–18041
15. Ulvmar MH, Werth K, Braun A, Kelay P, Hub
E, Eller K, Chan L, Lucas B, Novitzky-Basso I,
Nakamura K, Rulcke T, Nibbs RJ, Worbs T,
Forster R, Rot A (2014) The atypical chemokine receptor CCRL1 shapes functional
CCL21 gradients in lymph nodes. Nat
Immunol 15(7):623–630
16.Okada T, Cyster JG (2007) CC chemokine
receptor 7 contributes to Gi-dependent T cell

motility in the lymph node. J Immunol
178(5):2973–2978
17. Cyster JG (2005) Chemokines, sphingosine-­1-­
phosphate, and cell migration in secondary
lymphoid organs. Annu Rev Immunol
23:127–159

18.Park SM, Angel CE, McIntosh JD, Books
AES, Middleditch M, Chen CJJ, Ruggiero K,
Cebon J, Dunbar PR (2014) Spingosine-1phosphate lyase is expressed by CD68+ cells
on the parenchymal side of marginal reticular
cells in human lymph nodes. Eur J Immunol
44:2425–2436
19.Garside P, Brewer JM (2008) Real-time imaging of the cellular interactions underlying tolerance, priming, and response to infection.
Immunol Rev 221:130–146
20.Ehrlich LI, Oh DY, Weissman IL, Lewis RS
(2009) Differential contribution of chemotaxis
and substrate restriction to segregate of immature and mature thymocytes. Immunity
31(6):986–998
21. Le Borgne M, Ladi E, Dzhagalov I, Herzmark
P, Liao YF, Chakraborty AK, Robey EA (2009)
The impact of negative selection on thymocyte
migration in the medullar. Nat Immunol
10(8):823–830
22. Richardson DS, Lichtman JW (2015) Clarifying
tissue clearing. Cell 162(2):246–257
23.Kumar V, Scandella E, Danuser R, Onder L,
Nitschke M, Fukui Y, Halin C, Ludewig B,
Stein JV (2010) Global lymphoid tissue remodelling during a viral infection is orchestrated by
a B cell-lymphotoxin-dependent pathway.

Blood 115(23):4725–4733

24.Germain RN, Meier-Schellersheim M, Nita-­
Lazar A, Fraser ID (2011) Systems biology in
immunology: a computational modelling perspective. Annu Rev Immunol 29:527–585
25. Kislitsyn A, Savinkov R, Novkovic M, Onder L,
Bocharov G (2015) Computational approach


8

Mark C. Coles

to 3D modeling of the lymph node geometry.
Computation 3(2):222–234
26.Ganesan A, Levchenko A (2012) Principles of
model building: an experimentation-aided
approach to development of models for signaling networks. Methods Cell Biol 110:1–17

27.Novkovic M, Onder L, Cupovic J, Abe J,
Bomze D, Cremasco V, Scandella E, Stein JV,
Bocharov G, Turley SJ, Ludewig B (2016)
Topological small-world organization of the
fibroblastic reticular cell network determines
lymph node functionality. PLoS Biol
14(7):e1002515

28. Garin A, Meyer-Hermann M, Contie M, Figge
MT, Buatois V, Gunzer M, Toellner KM, Elson
G, Kosco-Vilbois MH (2010) Toll-like receptor 4 signaling by follicular dendritic cells is

pivotal for germinal center onset and affinity
maturation. Immunity 33:84–95

29.Zhang Y, Meyer-Hermann M, George LA,
Figge MT, Khan M, Goodall M, Young SP,
Reynolds A, Falciani F, Waisman A, Notley
CA, Ehrenstein MR, Kosco-Vibois M, Toeliner
KM (2013) Germinal center B cells govern
their own fate via antibody feedback. J Exp
Med 210:457–464


Chapter 2
Analysis of Thymocyte Migration, Cellular Interactions,
and Activation by Multiphoton Fluorescence Microscopy
of Live Thymic Slices
Jessica N. Lancaster and Lauren I.R. Ehrlich
Abstract
Thymocytes migrate through discrete compartments within the thymus, engaging in cellular interactions
essential for their differentiation into functional and self-tolerant T cells. Thus, understanding the temporal and spatial behavior of thymocytes within an intact thymic microenvironment is critical for elucidating
processes governing T cell development. Towards this end, we describe methods for preparing thymic
explant slices, in which the migration of thymocytes through three-dimensional space can be probed using
time-lapse, multiphoton fluorescence microscopy. Thymocytes, enriched for developmental subsets of
interest, are labeled with cytoplasmic fluorescent dyes, and seeded onto live thymic slices that express an
endogenous, stromal cell-specific fluorescent reporter. In response to chemotactic cues produced by
thymic stromal cells, the labeled thymocytes migrate within thymic microenvironments and engage in
cellular interactions that recapitulate a physiological system, which can be readily imaged. Here we describe
specimen preparation that maintains the integrity of thymic structures. We also describe imaging protocols
for acquiring multiple fluorochrome channels to enable detection of thymocyte:stromal cell interactions
and quantification of relative intracellular calcium levels to monitor T cell receptor activation. Parameters

for quantifying motility and interaction behaviors during data analysis are also briefly described. The thymic slice is a versatile tool for probing live cell behaviors and developing novel hypotheses not readily
apparent by static experimental methods.
Key words Multiphoton fluorescence microscopy, Thymocyte, Thymus, Migration, Cell–cell interaction, Calcium flux, TCR activation

1  Introduction
Sequential stages of T cell maturation are coordinated with ordered
migration through discrete compartments of the thymus [1, 2].
The diverse stromal cells within the thymus orchestrate the localization and timing of thymocyte movement through different thymic regions in order to provide signals for thymocyte survival,
proliferation, and differentiation [3]. Thymocytes respond to
stromal-­derived migratory cues through the regulated expression
of chemokine receptors and integrins, and in turn engage stromal
George Edward Rainger and Helen M. Mcgettrick (eds.), T-Cell Trafficking: Methods and Protocols, Methods in Molecular Biology,
vol. 1591, DOI 10.1007/978-1-4939-6931-9_2, © Springer Science+Business Media LLC 2017

9


10

Jessica N. Lancaster and Lauren I.R. Ehrlich

cells in crosstalk that modulates stromal cell differentiation [4].
Thus, describing the migratory behavior of thymocytes is essential
for understanding T cell development. Though the importance of
thymocyte migration in distinct thymic microenvironments is well
appreciated, the molecular cues that drive thymocyte localization
and interactions with stromal cells are not completely understood.
Insights into these mechanisms are often inaccessible using standard immunological techniques, which either offer snapshots of
cellular processes or fail to recapitulate processes that occur only in
organized, three-dimensional tissues. Mechanisms governing thymocyte localization or interactions with stromal cells that promote

thymocyte differentiation or selection can be queried by quantifying parameters of thymocyte migration as it occurs in situ.
Two-photon/multiphoton microscopy enables the visualization of fluorescent structures within tissues, due to a nonlinear
excitation phenomenon that results in decreased light scattering
and improved optical sectioning [5]. Thus, multiphoton microscopy is a valuable tool for observing fluorescently labeled live cells
as they migrate within intact tissue. However, imaging within the
thymus poses technical challenges: because the thymus is located
next to the heart, intravital imaging is subject to motion artifacts
[6]. In addition, central medullary regions are buried in the center
of intact thymic lobes, where they are often at the detection limit
of multiphoton resolution [7]. In order to circumvent these issues,
techniques have been developed to generate live slices of thymus
that are maintained under physiological conditions [6]. By the
introduction of both exogenous and genetically encoded fluorescent markers into thymocyte and stromal cells in the system, the
migration and behavior of thymocytes can be visualized by live cell
imaging within the intact thymic microenvironment [7, 8].
Although thymic slices are tissue explants, thymocytes within slices
remain responsive to chemotactic cues released by thymic stroma,
localize to the thymic microenvironments appropriate for distinct
stages of differentiation [8], and retain motility comparable to thymocytes migrating in intact thymic lobes explants [7]. Chemotactic
responsiveness and thymocyte migration occur in both human and
mouse thymic slices [9]. Positive and negative selection of thymocytes, which are critical checkpoints governing development of
functional, self-tolerant T cells, are also supported on thymic slices
[6, 7, 10]. Thus, the thymic slice has proven to be invaluable in the
application of multiphoton microscopy to study the motility, localization, and stromal interactions of thymocyte subsets.
Positive and negative selection are driven by activation of T cell
receptors (TCRs) on thymocytes, which is induced by ligation of
self-peptide:major histocompatibility complex molecules on thymic antigen presenting cells (APCs). The concentration of
­intracellular calcium, a secondary messenger downstream of TCR
activation, has been shown to rapidly increase and fall within

Multi-photon Microscopy of Thymocyte Migration

11

seconds of TCR activation [11] and can therefore serve as a proxy
for TCR signaling. With the availability of many calcium-sensitive
fluorescent indicators, live cell tracking of intracellular calcium has
become an important tool in studying T cell activation [11, 12].
Cell-permeant dyes, such as Indo1 and Fluo-3/4/5, have altered
fluorescence emission properties upon binding intracellular calcium [13, 14]. Chameleon is a genetically encoded fluorescent
protein, whose fluorescence depends on calcium-dependent binding [15]. Among these indicators, leak-resistant Indo1, also known
as Indo-PE3, has been used in imaging thymic slices, due to ease
of loading and relatively bright intensity [6]. In addition, Indo1
emission is ratiometric, with intensity at shorter wavelengths
increasing at higher calcium concentrations, while intensity at longer wavelengths decreases concomitantly, allowing it to serve as its
own internal fluorescence control. Previous studies that quantified
intracellular calcium in thymocytes undergoing selection demonstrated that thymocytes exhibit reduced motility upon calcium flux
[6] and form aggregates with elevated calcium levels [16]. Further,
the strength and frequency of calcium signaling have been used to
distinguish positive and negative selection events [17]. Transient
calcium fluxes, induced by relatively low avidity ligands, are integrated during positive selection [18], while negative selection is
marked by sustained calcium elevation induced by high avidity
ligands [19]. Thus, multiphoton imaging of intracellular calcium
has advanced the study of thymocyte activation as it occurs in real
time within the thymus.
This chapter serves as a detailed protocol of thymic slice generation. Here we describe how isolated thymocyte subsets can be
labeled and introduced into thymic slices for the purpose of multiphoton imaging. Multiphoton imaging of thymic slices can yield
novel insights into the molecular and cellular mechanisms that
govern

real-time
behavior
of
thymocyte
migration,
thymocyte:stromal cell interactions, and TCR activation.

2  Materials
2.1  Preparation
of Thymocytes

1.Mice: Select strain based on thymocyte subset or molecule of
interest. For example, Rag2−/− mice can be used to image thymocytes blocked at an early maturation stage [20]; chemokine
receptor-deficient strains such as Ccr7−/− can be used to study
the role of CCR7 on thymocyte motility [21]; and TCR
­transgenics, such as OT-I mice [22], can be used to study
selection of thymocytes with a defined TCR specificity.
2.
Rodent euthanasia: CO2 gas and chamber (VetEquip,
Livermore, CA).
3.Spray bottle of 10% ethanol in water.
4.Paper towels.


12

Jessica N. Lancaster and Lauren I.R. Ehrlich

5.Dissection instruments: surgical scissors (Roboz RS-5910),
fine spring scissors (Roboz RS-5668), angled surgical scissors

(Roboz RS-5918), forceps (Roboz 5), angled forceps (Roboz
5/45), curved forceps (Roboz RS-5135).
6.40 μm nylon mesh cell strainer.
7.BrightLine hemacytometer.
8.Trypan blue.
9.Brightfield tissue culture microscope.
10.Fluorescent probes: CellTracker dyes (Molecular Probes) are
cytoplasmic loading dyes available in a wide range of colors;
Indo1AM is a cell-permeant, leak-resistant ratiometric calcium
indicator dye.
11.Rat anti-mouse antibodies for depletion of hematopoietic lineages, e.g., anti-B220 (clone RA3.3A1/6.1), anti-Ter119
(BE0183), anti-Gr1 (RB6-8C5), anti-CD11b (M1/70); or
thymocyte subsets, e.g., anti-CD3 (17A2), anti-CD4 (GK1.5),
anti-CD8 (53.6.72), anti-CD25 (PC-61.5.3).
12. DNYNALTM Dynabeads sheep anti-rat (Invitrogen) magnetic
beads.
13.Dyna-Mag 15 magnet.
14.DRPMI medium: powdered RPMI 1640 medium deficient in
phenol red, sodium bicarbonate, and l-glutamine, supplemented with 0.2 g/L sodium bicarbonate and 20 mM HEPES.
15.DRPMI with 10% bovine calf serum (BCS).
16.Phosphate buffered saline (PBS).
17.PBS with 2% BCS.
18.Complete RPMI medium: RPMI 1640 medium, supplemented with 2 mM L-glutamine, 50 U/mL penicillin, 50 mg/
mL streptomycin, and 10% (v/v) fetal bovine serum (FBS).
2.2  Generation
of Thymic Slices

1. Mice: 3–4 weeks of age; select strain based on thymic microenvironment and endogenous fluorescent reporters of interest.
For example, in RIP-mOVA mice, medullary APCs express
ovalbumin as a TRA [23], and can thus be used to study negative selection of OT-I TCR transgenic thymocytes; AireEGFP

knockin mice express green fluorescent protein (GFP) in AIRE+
medullary thymic epithelial cells [24]; MaFIA express GFP in
Csf1r-expressing macrophages [25]; and CD11c-EYFP express
yellow fluorescent protein in CD11c+ dendritic cells [26].
2.Rodent anesthesia: Isoflurane (Southmedic, Ontario, CA) and
vaporizer chamber (VetEquip).
3. Rodent guillotine (World Precision Instruments, Sarasota, FL,
cat. no. DCAP or similar).
4.60 × 15 mm tissue culture dish.


Multi-photon Microscopy of Thymocyte Migration

13

5.BioLite 35 mm tissue culture dish.
6.PTFE-coated stainless steel double-edged razor blade.
7.5 mL volume plastic specimen cups.
8.Superglue.
9.Tapered end micro spatula, spoon spatula.
10.4% (w/v) low melting point agarose in PBS.
11.T-type thermocouple with thin flexible probe.
12. VT-1000 vibratome.
2.3  Seeding
of Labeled
Thymocytes
into Thymic Slices

1.Millicell 0.4 μm, 30 mm diameter cell culture membrane
inserts (EMD Millipore).

2.4  Acquisition
by Multiphoton
Microscopy

1.Upright fluorescence microscope with detector array, fluorescence emission filter sets, water immersion objective lens,
titanium:sapphire laser for multiphoton excitation, and laser
scanning system. Our setup employs the PrairieView Ultima
IV (Bruker, Billerica, MA), 20× NA 0.95 Plan Fluor water
immersion objective (Olympus, Tokyo, Japan), photomultiplier tube (PMT) detectors, 400/50, 480/40, 535/50, and
607/45 bandpass emission filters (Chroma Technology,
Bellows Falls, VT), and two MaiTai HP lasers (SpectralPhysics,
Santa Clara, CA).

2.Silica grease.

2.Image acquisition software: microscope vendor specific, our
setup employs PrairieView (Bruker).
3.Heated stage chamber RC-26GLP (Warner Instruments,
Hamden, CT).
4.Inline perfusion heater SH-27B (Warner Instruments).
5.Nylon specimen harp SHD-26GH/2 (Warner Instruments).
6.Perfusion 300 mL IV set and flow regulator (Wolf Medical
Supply, Sunrise, FL, cat. no. RF5600).
7.Bubbling stone and PVC tubing (Warner Instruments).
8.95% oxygen with 5% carbon dioxide.
9.Imaging perfusion medium: powdered RPMI 1640 medium
deficient in phenol red, sodium bicarbonate, and l-glutamine,
supplemented with 2 g/L sodium bicarbonate, and 5 mM
HEPES. Adjust pH to 7.4, then add 0.85 mM calcium chloride for a final calcium concentration of 1.25 mM.

2.5  Image Analysis
of Slices

1.Imaging data analysis software: Analysis programs vary in
accessibility of the user interface and price. For our analysis we
employ Fiji/ImageJ (National Institutes of Health, Bethesda,
MD), Imaris version 8.2.0 (Bitplane, Concord, MA), and
MATLAB version R2015a (Mathworks, Natick, MA).


14

Jessica N. Lancaster and Lauren I.R. Ehrlich

3  Methods
3.1  Preparation
of Thymocytes
3.1.1  Harvest
Thymocytes

1. Sacrifice the mouse in CO2 chamber and confirm euthanasia by
secondary means such as cervical dislocation.
2.Secure the mouse to the dissection board in the supine position, with its abdomen exposed, by pinning down the appendages. Moisten fur with 10% ethanol to keep the fur clear of the
dissection.
3.Begin dissection of the mouse by lifting the skin up at the
abdomen using curved forceps, and cut the skin up the midline
from abdomen to throat using scissors.
4.Make a perpendicular cut across the upper abdomen, and pull
the skin away from the thin subdermal tissue layer. Sliding the
scissor blades between the skin and subdermal layer will disconnect the fascia holding the tissues together.

5.Lift the subdermal tissue using curved forceps and cut just
below the ribs, taking care not to cut any internal organs, then
extend the cut along the base of the ribs.
6.Pierce the diaphragm with scissors, taking care not to cut the
liver or lungs.
7.Cut superiorly through the ribs on both lateral sides, ending
near the axilla. Using forceps lift up the front of the ribcage,
exposing the heart and thymus above it.
8. Use the curved forceps to pull the thymus out of the chest cavity by pulling at its base.
9.Gently rinse the thymus by dipping once a beaker of PBS to
wash off residual red blood cells.
10. Prepare a single cell suspension of thymocytes by mechanically
dissociating thymic tissue through a cell strainer into 5 mL of
DRPMI with 10% BCS.
11.Count the live cells under a brightfield microscope by placing
a 1:1 mixture of cell suspension and Trypan Blue on a hemacytometer slide. Live cells will be round and transparent, while
dead cells are stained blue. Calculate the concentration of cells
per the hemacytometer manufacturer instructions.

3.1.2  Enrich the Desired
Thymocyte Subset

1.Resuspend thymocytes at 2 × 108 cells/mL in PBS with 2%
BCS.
2.Depending on which thymocyte subset is needed for the
experiment, thymocytes are then incubated with purified rat
anti-mouse antibodies specific for cell surface markers present
on lineages to be depleted. For example, to enrich for CD4
single positive thymocytes, we incubate thymocytes with anti-­
CD8 (96 μg/mL), and antibodies against lineage markers

Multi-photon Microscopy of Thymocyte Migration

15

CD25, CD11b, Gr1, Ter119, and B220 (5 μg/mL for each);
similarly, we enrich for CD8 single positive thymocytes with
anti-CD4 (21 μg/mL) and the same lineage cocktail.
3.Incubate the cells in antibodies on ice for 30 min, and then
wash twice by resuspending in 5 mL of PBS with 2% BCS, pelleting the cells in swinging bucket centrifuge (5 min at 163 × g),
and discarding the supernatant.
4.Prepare sheep anti-rat magnetic beads for use at a ratio of 2:1
cells:beads by washing the appropriate number of beads twice
in 1 mL of PBS with 2% BCS, separating the beads from the
wash by attaching the tube to the magnetic holder and pipetting out the supernatant with a P1000 pipet. Resuspend in 1
mL of PBS with 2% BCS.
5.Resuspend cells at 7 × 106/mL in PBS with 2% BCS with
beads.
6.Incubate at room temperature on a rocking platform for
10 min.
7. Place the tube on the magnetic holder and collect the unbound
supernatant using a P1000 pipet.
8.Repeat magnetic depletion with a second round of freshly
washed beads for a ratio of 4:1 cells:beads, based on the original cell count.
9.Count the depleted thymocytes (as in Subheading 3.1.1,
step k).
3.1.3  Label Thymocytes
with a Fluorescent Probe

1. Distribute 106–107 cells each into 1.5 mL tubes, such that cells
from one tube will be seeded onto one slice.
2.Prepare a solution of the desired fluorescent dye by vigorously
mixing the dye in DRPMI medium (see Note 1). For example,
thymocytes can be imaged in the 607 nm emission channel by
staining them with 2 μM CellTracker CMTPX Red in 1.5 mL
DRPMI prewarmed to 37 °C; calcium imaging can be recorded
in the 400 nm and 480 nm emission channels by staining with
2 μM Indo1AM in 1.5 mL prewarmed DRPMI.
3.Pellet the cells in a fixed angle centrifuge (468 × g), aspirate
supernatant, and resuspend the thymocytes thoroughly in 1.5
mL staining solution.
4.Incubate at 37 °C in a water bath.
5.After 30 min, pellet the cells using a fixed angle centrifuge
(468 × g), aspirate supernatant, and resuspend in 1.5 mL complete RPMI medium.
6.Incubate at 37 °C in a water bath for 30 min to allow excess
dye to destain.