Methods in
Molecular Biology 1584

Cosima T. Baldari
Michael L. Dustin Editors

The Immune
Synapse
Methods and Protocols


Methods

in

Molecular Biology

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

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The Immune Synapse
Methods and Protocols

Edited by

Cosima T. Baldari
Department of Life sciences, University of Siena, Siena, Siena, Italy

Michael L. Dustin
University of Oxford, Kennedy Institute of Rheumatology, Headington, Oxford, UK


Editors
Cosima T. Baldari
Department of Life sciences
University of Siena
Siena, Siena, Italy

Michael L. Dustin
University of Oxford, Kennedy Institute
of Rheumatology
Headington, Oxford, UK

ISSN 1064-3745    ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-6879-4    ISBN 978-1-4939-6881-7 (eBook)
DOI 10.1007/978-1-4939-6881-7
Library of Congress Control Number: 2017931687
© Springer Science+Business Media LLC 2017
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Preface
Initiation of the T cell-mediated adaptive immune response to pathogens is crucially dependent on the assembly of a highly specialized signaling platform that forms at the interface
of a T cell and an antigen-presenting cell (APC) bearing specific peptide antigen associated
with major histocompatibility molecules, known as the immune synapse. From its initial
description as a membrane domain characterized by the segregation in concentric subdomains of specific receptors that is accompanied by the polarization of the microtubule-­
organizing center towards the APC contact area, our understanding of the structure,
dynamics, and function of the immune synapse has rapidly evolved. It is now clear that the
mature bull’s eye synapse marks the final phase of an extremely dynamic process where
microclusters of receptors and signaling mediators converge as they signal towards the center of the IS, where they are either internalized to be targeted for degradation or released
as microvesicles to convey information and instructions to the APC. Vesicular traffic has
emerged as a central player in ensuring not only polarized delivery of cytokines and enzymes
to target cells by T cell effectors but also sustained signaling at the immune synapse and
modulation of the APC during naive T cell activation. Moreover the T cell immune synapse
has recently emerged as a paradigm for a variety of immune cell interactions that include
synapses formed by B cells, NK, and mast cells. The remarkable progress in this rapidly
moving area has required the development of powerful techniques and tools of analysis,
ranging from super-resolution microscopy and electron tomography, to the generation of
highly specific micropatterned surfaces for studying the dynamics of microclusters and single molecules, to a variety of molecular probes to image signaling dynamics, to the imaging
of immune cell interactions in vivo, to robust computational methods to address the spatiotemporal complexity of the immune synapse. This book has collected all the essential

protocols that are currently used to study the immune synapse, addressing (1) methods for
the study of the dynamics of immune synapse assembly; (2) methods for the study of
vesicular traffic at the immune synapse; (3) new high resolution imaging, biophysical, and
computational methods for the study of the immune synapse; (4) methods for the study of
effector immune synapses; (5) methods for the study of B cell, NK, and mast cell immune
synapses; and (6) methods for the study of immune interactions in vivo. This timely and
exhaustive collection of protocols is expected to be of interest to immunologists and, at a
more general level, to cell biologists, biophysicists, and computational biologists.
Siena, Italy
Headington, Oxford, UK

Cosima T. Baldari
Michael L. Dustin

v


Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
  1 The Immune Synapse: Past, Present, and Future . . . . . . . . . . . . . . . . . . . . . . .
Michael L. Dustin and Cosima T. Baldari
  2 Analyzing Actin Dynamics at the Immunological Synapse . . . . . . . . . . . . . . . . .
Katarzyna I. Jankowska and Janis K. Burkhardt
  3 Analysis of Microtubules and Microtubule-Organizing Center
at the Immune Synapse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Noelia Blas-Rus, Eugenio Bustos-Morán, Francisco Sánchez-Madrid,
and Noa B. Martín-Cófreces
  4 Analyzing the Dynamics of Signaling Microclusters . . . . . . . . . . . . . . . . . . . . .

Akiko Hashimoto-Tane, Tadashi Yokosuka, and Takashi Saito
  5 Reconstitution of TCR Signaling Using Supported Lipid Bilayers . . . . . . . . . . .
Xiaolei Su, Jonathon A. Ditlev, Michael K. Rosen, and Ronald D. Vale
  6 Plasma Membrane Sheets for Studies of B Cell Antigen Internalization
from Immune Synapses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Carla R. Nowosad and Pavel Tolar
  7 Studying the Dynamics of TCR Internalization at the Immune Synapse . . . . . .
Enrique Calleja, Balbino Alarcón, and Clara L. Oeste
  8 T Cell Receptor Activation of NF-κB in Effector T Cells: Visualizing
Signaling Events Within and Beyond the Cytoplasmic Domain
of the Immunological Synapse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Maria K. Traver, Suman Paul, and Brian C. Schaefer
  9 Imaging Vesicular Traffic at the Immune Synapse . . . . . . . . . . . . . . . . . . . . . . .
Jérôme Bouchet, Iratxe del Río-Iñiguez, and Andrés Alcover
10 Analysis of TCR/CD3 Recycling at the Immune Synapse . . . . . . . . . . . . . . . . .
Laura Patrussi and Cosima T. Baldari
11 Simultaneous Membrane Capacitance Measurements and TIRF
Microscopy to Study Granule Trafficking at Immune Synapses . . . . . . . . . . . . .
Marwa Sleiman, David R. Stevens, and Jens Rettig
12 Mathematical Modeling of Synaptic Patterns . . . . . . . . . . . . . . . . . . . . . . . . . .
Anastasios Siokis, Philippe A. Robert, and Michael Meyer-Hermann
13 Super-resolution Analysis of TCR-Dependent Signaling: Single-Molecule
Localization Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Valarie A. Barr, Jason Yi, and Lawrence E. Samelson
14 Förster Resonance Energy Transfer to Study TCR-pMHC Interactions
in the Immunological Synapse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Gerhard J. Schütz and Johannes B. Huppa

vii


1
7

31

51
65

77
89

101
129
143

157
171

183

207


viii

Contents

15 Two-Dimensional Analysis of Cross-Junctional Molecular Interaction
by Force Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
Lining Ju, Yunfeng Chen, Muaz Nik Rushdi, Wei Chen,

and Cheng Zhu
16 Studying Dynamic Plasma Membrane Binding of TCR-CD3 Chains
During Immunological Synapse Formation Using Donor-Quenching
FRET and FLIM-FRET . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259
Etienne Gagnon, Audrey Connolly, Jessica Dobbins,
and Kai W. Wucherpfennig
17 Revealing the Role of Microscale Architecture in Immune Synapse
Function Through Surface Micropatterning . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
Joung-Hyun Lee and Lance C. Kam
18 Spatial Control of Biological Ligands on Surfaces Applied
to T Cell Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
Haogang Cai, David Depoil, James Muller, Michael P. Sheetz,
Michael L. Dustin, and Shalom J. Wind
19 Probing Synaptic Biomechanics Using Micropillar Arrays . . . . . . . . . . . . . . . . . 333
Weiyang Jin, Charles T. Black, Lance C. Kam, and Morgan Huse
20 Microchannels for the Study of T Cell Immunological Synapses and Kinapses . . . 347
Hélène D. Moreau, Philippe Bousso, and Ana-Maria Lennon-Duménil
21 Purification of LAT-Containing Membranes from Resting
and Activated T Lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355
Claire Hivroz, Paola Larghi, Mabel Jouve, and Laurence Ardouin
22 Quantitative Phosphoproteomic Analysis of T-Cell Receptor Signaling . . . . . . . 369
Nagib Ahsan and Arthur R. Salomon
23 Imaging Asymmetric T Cell Division . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
Mirren Charnley and Sarah M. Russell
24 Ultrastructure of Immune Synapses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
Jaime Llodrá
25 Systems Imaging of the Immune Synapse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409
Rachel Ambler, Xiangtao Ruan, Robert F. Murphy,
and Christoph Wülfing
26 Comprehensive Analysis of Immunological Synapse Phenotypes

Using Supported Lipid Bilayers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423
Salvatore Valvo, Viveka Mayya, Elena Seraia, Jehan Afrose,
Hila Novak-Kotzer, Daniel Ebner, and Michael L. Dustin
27 Studying Immunoreceptor Signaling in Human T Cells
Using Electroporation of In Vitro Transcribed mRNA . . . . . . . . . . . . . . . . . . . 443
Omkar Kawalekar, Carl H. June, and Michael C. Milone
28 A Protein Expression Toolkit for Studying Signaling in T Cells . . . . . . . . . . . . . 451
Ana Mafalda Santos, Jiandong Huo, Deborah Hatherley, Mami Chirifu,
and Simon J. Davis
29 Imaging the Effector CD8 Synapse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473
Gordon L. Frazer, Yukako Asano, and Gillian M. Griffiths


Contents

30 The Mast Cell Antibody-Dependent Degranulatory Synapse . . . . . . . . . . . . . .
Salvatore Valitutti, Régis Joulia, and Eric Espinosa
31 Measurement of Lytic Granule Convergence After Formation
of an NK Cell Immunological Synapse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Hsiang-Ting Hsu, Alexandre F. Carisey, and Jordan S. Orange
32 Studying the T Cell-Astrocyte Immune Synapse . . . . . . . . . . . . . . . . . . . . . . . .
George P. Cribaro, Elena Saavedra-López, Paola V. Casanova,
Laura Rodríguez, and Carlos Barcia
33 Aberrant Immunological Synapses Driven by Leukemic
Antigen-Presenting Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Fabienne McClanahan Lucas and John G. Gribben
34 Studying the Immune Synapse in HIV-1 Infection . . . . . . . . . . . . . . . . . . . . . .
Iratxe del Río-Iñiguez, Jérôme Bouchet, and Andrés Alcover
35 In Vivo Imaging of T Cell Immunological Synapses and Kinapses
in Lymph Nodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Hélène D. Moreau and Philippe Bousso
36 Studying Dendritic Cell-T Cell Interactions Under In Vivo Conditions . . . . . .
Nicholas van Panhuys

ix

487

497
517

533
545

559
569

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585


Contributors
Jehan Afrose  •  University of Oxford, Oxford, UK
Nagib Ahsan  •  Brown University and Rhode Island Hospital, Providence, USA
Balbino Alarcón  •  Universidad Autónoma de Madrid, Madrid, Spain
Andrés Alcover  •  Institut Pasteur, Paris, France
Rachel Ambler  •  University of Bristol, Bristol, UK
Laurence Ardouin  •  Institut Curie, Paris, France
Yukako Asano  •  Cambridge Institute for Medical Research, Cambridge, UK
Cosima T. Baldari  •  University of Siena, Siena, Italy
Carlos Barcia  •  Universidad Autónoma de Barcelona, Barcelona, Spain

Valerie A. Barr  •  National Cancer Institute, Bethesda, USA
Charles T. Black  •  Brookhaven National Laboratory, New York, USA
Noelia Blas-Rus  •  Universidad Autónoma de Madrid, Madrid, Spain
Jérôme Bouchet  •  Institut Pasteur, Paris, France
Philippe Bousso  •  Institut Pasteur, Paris, France
Janis K. Burkhardt  •  University of Pennsylvania, Philadelphia, USA
Eugenio Bustos-Morán  •  Centro Nacional Investigaciones Cardiovasculares (CNIC),
Madrid, Spain
Haogang Cai  •  Columbia University, New York, USA
Enrique Calleja  •  Universidad Autónoma de Madrid, Madrid, Spain
Alexandre F. Carisey  •  Texas Children’s Hospital and Baylor College of Medicine,
Houston, USA
Paola V. Casanova  •  Universidad Autónoma de Barcelona, Barcelona, Spain
Mirren Charnley  •  Swinburne University of Technology, Hawthorn, VIC, Australia;
Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia
Yunfeng Chen  •  Institute of Technology, Atlanta, USA
Wei Chen  •  Zhejiang University, Hangzhou, Zhejiang, China
Mami Chirifu  •  University of Oxford, Oxford, UK
Audrey Connolly  •  University of Montreal, Montreal, Canada
George P. Cribaro  •  Universidad Autónoma de Barcelona, Barcelona, Spain
Simon J. Davis  •  University of Oxford, Oxford, UK
Iratxe del Río-Iñiguez  •  Institut Pasteur, Paris, France
David Depoil  •  University of Oxford, Oxford, UK
Jonathon A. Ditlev  •  Marine Biological Laboratory, Woods Hole, USA; University of Texas,
Texas, USA
Jessica Dobbins  •  Dana-Farber Cancer Institute and Harvard Medical School, Boston, USA
Michael L. Dustin  •  University of Oxford, Oxford, UK; New York University School
of Medicine, New York, USA
Daniel Ebner  •  University of Oxford, Oxford, UK
Eric Espinosa  •  University of Toulouse, Toulouse, France

Gordon L. Frazer  •  Cambridge Institute for Medical Research, Cambridge, UK
Etienne Gagnon  •  University of Montreal, Montreal, Canada

xi


xii

Contributors

John G. Gribben  •  Queen Mary University of London, London, UK
Gillian M. Griffiths  •  Cambridge Institute for Medical Research, Cambridge, UK
Akiko Hashimoto-Tane  •  RIKEN Center for Integrative Medical Sciences, Yokohama, Japan
Deborah Hatherley  •  University of Oxford, Oxford, UK
Claire Hivroz  •  Institut Curie, Paris, France
Hsiang-Ting Hsu  •  Texas Children’s Hospital and Baylor College of Medicine, Houston, USA
Jiandong Huo  •  University of Oxford, Oxford, UK
Johannes B. Huppa  •  Technical University of Vienna, Vienna, Austria
Morgan Huse  •  Memorial Sloan-Kettering Cancer Center, New York, USA
Katarzyna I. Jankowska  •  University of Pennsylvania, Philadelphia, USA
Weiyang Jin  •  Columbia University, New York, USA
Régis Joulia  •  University of Toulouse, Toulouse, France
Mabel Jouve  •  Institut Curie, Paris, France
Lining Ju  •  University of Sydney, Sydney, Australia
Carl H. June  •  University of Pennsylvania, Philadelphia, PA, USA
Lance C. Kam  •  Columbia University, New York, USA
Omkar Kawalekar  •  University of Pennsylvania, Philadelphia, PA, USA
Paola Larghi  •  University of Milan, Milan, Italy; Istituto Nazionale Genetica Molecolare,
‘Romeo ed Enrica Invernizzi’, INGM, Milan, Italy
Joung-Hyun Lee  •  Columbia University, New York, USA

Ana-Maria Lennon-Duménil  •  Institut Curie, PSL Research University, Paris, France
Jaime Llodrá  •  University of Bern, Bern, Switzerland
Noa B. Martín-Cófreces  •  Universidad Autónoma de Madrid, Madrid, Spain
Viveka Mayya  •  University of Oxford, Oxford, UK
Fabienne McClanahan Lucas  •  Queen Mary University of London, London, UK; The
Ohio State University, Columbus, OH, USA
Michael Meyer-Hermann  •  Helmholtz Centre for Infection Research, Braunschweig,
Germany
Michael C. Milone  •  University of Pennsylvania, Philadelphia, PA, USA
Hélène D. Moreau  •  Institut Curie, PSL Research University, Paris, France; Institut
Pasteur, Paris, France
James Muller  •  New York University School of Medicine, New York, USA
Robert F. Murphy  •  Carnegie Mellon University, Pittsburgh, USA
Hila Novak-Kotzer  •  Kennedy Institute of Rheumatology, Nuffield Department of
Orthopedics, Rheumatology and Musculoskeletal Sciences, The University of Oxford,
Oxford, UK
Carla R. Nowosad  •  Francis Crick Institute, London, UK
Clara L. Oeste  •  Universidad Autónoma de Madrid, Madrid, Spain
Jordan S. Orange  •  Texas Children’s Hospital and Baylor College of Medicine, Houston, USA
Laura Patrussi  •  University of Siena, Siena, Italy
Suman Paul  •  Uniformed Services University, Bethesda, USA
Jens Rettig  •  Universität des Saarlandes, Homburg/Saar, Germany
Philippe A. Robert  •  Helmholtz Centre for Infection Research, Braunschweig, Germany;
Université Montpellier II, Montpellier, France
Laura Rodríguez  •  Universidad Autónoma de Barcelona, Barcelona, Spain
Michael K. Rosen  •  Marine Biological Laboratory, Woods Hole, USA; University of Texas,
Texas, USA
Xiangtao Ruan  •  Carnegie Mellon University, Pittsburgh, USA



Contributors

xiii

Muaz Nik Rushdi  •  Institute of Technology, Atlanta, USA
Sarah M. Russell  •  Swinburne University of Technology, Hawthorn, VIC, Australia;
Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia; University
of Melbourne, Parkville, VIC, Australia
Elena Saavedra-López  •  Universidad Autónoma de Barcelona, Barcelona, Spain
Takashi Saito  •  RIKEN Center for Integrative Medical Sciences, Yokohama, Japan
Arthur R. Salomon  •  Brown University and Rhode Island Hospital, Providence, USA
Lawrence E. Samelson  •  National Cancer Institute, Bethesda, USA
Francisco Sánchez-Madrid  •  Universidad Autónoma de Madrid, Madrid, Spain
Ana Mafalda Santos  •  University of Oxford, Oxford, UK
Brian C. Schaefer  •  Uniformed Services University, Bethesda, USA
Gerhard Schütz  •  TU Wien, Vienna, Austria
Elena Seraia  •  University of Oxford, Oxford, UK
Michael P. Sheetz  •  Columbia University, New York, USA; National University
of Singapore, Singapore, Singapore
Anastasios Siokis  •  Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany
Marwa Sleiman  •  Universität des Saarlandes, Homburg/Saar, Germany
David R. Stevens  •  Universität des Saarlandes, Homburg/Saar, Germany
Xiaolei Su  •  Marine Biological Laboratory, Woods Hole, USA; University of California
San Francisco, San Francisco, USA
Pavel Tolar  •  Francis Crick Institute, London, UK; Imperial College London, London, UK
Maria K. Traver  •  Uniformed Services University and Henry M Jackson Foundation for
the Advancement of Military Medicine, Bethesda, USA
Ronald D. Vale  •  Marine Biological Laboratory, Woods Hole, USA; University of
California, San Francisco, USA
Salvatore Valitutti  •  University of Toulouse, Toulouse, France

Salvatore Valvo  •  University of Oxford, Oxford, UK
Nicholas van Panhuys  •  Sidra Medical and Research Center, Doha, Qatar
Shalom J. Wind  •  Columbia University, New York, USA
Kai W. Wucherpfennig  •  Dana-Farber Cancer Institute, Boston, USA; Harvard Medical
School, Boston, USA
Christoph Wülfing  •  University of Bristol, Bristol, UK
Jason Yi  •  Columbia University, New York, USA
Tadashi Yokosuka  •  RIKEN Center for Integrative Medical Sciences, Yokohama, Japan
Cheng Zhu  •  Georgia Institute of Technology, Atlanta, USA


Chapter 1
The Immune Synapse: Past, Present, and Future
Michael L. Dustin and Cosima T. Baldari
Abstract
Immunological synapses are specialized cell-cell junctions characterized by (1) close apposition of the
immune cell membrane with the membrane of another cell driven by adaptive or innate immune recognition, (2) adhesion, (3) stability, and (4) directed secretion. This phenomenon was first recognized in the
1970s and the early 1980s through electron microscopy of ex vivo functioning immune cells. Progressive
advances in fluorescence microscopy and molecular immunology in the past 20 years have led to rapid
progress on understanding the modes of cell-cell interaction and underlying molecular events. This volume contains a diverse range of protocols that can be applied to the study of the immunological synapses
and related immune cell junctions both in vitro and in vivo; and in disease settings in animal models and
humans. We have also included chapters on critical molecular tools such as protein expression and mRNA
electroporation that underpin or expand imaging approaches, although they are not specific to the study
of immune synapses. We hope that these chapters will be of use to people entering the field as well as seasoned practitioners looking to expand their repertoire of methods.
Key words Science history, Fluorescence, Affinity, Modeling, Microscopy

1  Introduction
Phagocytosis and antibodies were described within a decade of each
other at the turn of the nineteenth to twentieth century [1, 2], but
while the direct physical role of the phagocyte in phagocytosis was

immediately evident, it took another 60 years to recognize that
lymphocytes made antibodies [3]. The existence of two types of
lymphocytes and the need for their cooperation in antibody production led to studies in the 1970s on the physical interaction of T
lymphocytes that accompanied T cell help and cytotoxicity [4]. The
role of macrophages and dendritic cells in the generation of T cell
help was discovered in this same period with the genetic evidence
for MHC restriction of T cell responses [5]. These studies initially
relied on electron microscopy to reveal the close membrane alignment between lymphocytes that was well described in solid organs
as representing adhesive junctions [6, 7]. The molecules that mediated the adhesion were discovered through function blocking
Cosima T. Baldari and Michael L. Dustin (eds.), The Immune Synapse: Methods and Protocols, Methods in Molecular Biology,
vol. 1584, DOI 10.1007/978-1-4939-6881-7_1, © Springer Science+Business Media LLC 2017

1


2

Michael L. Dustin and Cosima T. Baldari

monoclonal antibodies [8]. The field’s recognition of directed
secretion as a critical process in T cell cytotoxicity and the role of
cytoplasmic Ca2+ evaluation in the signaling process was synthesized
by Norcross as suggesting a “synaptic basic for T cell activation”
[9]. Seder and Paul further elaborated on this, summarizing 10 years
of work on T cell help for B cells and the role of directed secretion
of cytokines with specific label of “immunological synapse” in a
commentary in Cell [10]. Within a year of this, Kupfer presented
his first images of the supramolecular activation clusters that were
revealed by the application of wide-field fluorescence microscopy
with deconvolution to conjugates of T cells with B cell tumors [11].

Parallel work to address the measurement of 2D affinity using supported planar bilayers (SLB) advanced to reconstitution of T cell
activation with convergence on the time-dependent evolution of
the same pattern, which was defined as a mature immunological
synapse [12]. A reviewer of a predeceding paper that sought to
define an immunological synapse raised the caveat that “immunological synapse” was too broad a term to apply to a structure formed
by T cells, as other immune cells might use similar strategies [13].
This was of course correct and we can now consider the mature
immunological synapse to be the result of a common strategy
applied by many immune cell types that use immunoreceptors
including mast cells, multiple types of T cells, NK cells, B cells, neutrophils, macrophages, and dendritic cells [14–19]. In the subsequent years, the field has continued to evolve with technology and
many features of immunological synapses have been discovered,
including imaging of interaction of T cells and antigen presenting
cells in situ and in vivo [20–22]. The immunological synapse presents an outstanding opportunity in basic cell biology as T cells can
be triggered by well-defined inputs to display multiple modes of
motility and polarization [23–25]. The immunological synapse is
disrupted in primary immunodeficiency diseases [26, 27] and autoreactive T cells form defective immunological synapses [28]. The
immunological synapse concept has guided studies leading to lifesaving therapies, particularly in cancer immunotherapy [29, 30].
There are still many questions remaining and this book is meant to
provide a current and forward-­looking set of methods that will help
to address the next level of questions and allow further application
to improvement of human health.

2  Materials
This book is composed of 35 chapters (excluding this introductory
chapter) that present methods relevant to the characterization of
the immunological synapse. Some chapters present multiple proven
approaches to study a particular phenomenon within the immunological synapse or type of immunological synapse. Others present


The Immune Synapse: Past, Present, and Future


3

details of technical approaches that can be applied to the multiple
types of immunological synapses and other related biology. Yet
others present enabling technologies that are quite general in
applications in life sciences, such as methods for efficient expression of exogenous proteins in primary cells or recombinant proteins expression and purification. While of broad utility, we invited
them here because they are key enabling technologies for future
studies on immune synapses.
As a matter of MIMB style, the authors have been discouraged
from providing detailed information on suppliers for common
materials due to concerns about regional differences in chemical
supply. However, we have broken with this style in some instances
to identify suppliers for what appear to be common items (for
example, microscope coverslips or glass bottom 96-well plates)
when the authors have taken great effort to screen many potential
suppliers of similar items and identified particular sources that outperformed others in direct comparisons. These instances may be
further highlighted in the Notes section to describe the criteria for
selecting particular suppliers. This should be helpful in case any
reader has difficultly accessing particular suppliers in their regions.
The relevant screening criteria can be reapplied if necessary to find
a suitable alternative supplier. Furthermore, if individuals reading
these chapters run into problems with applying the protocols
included here, all of the authors are happy to be contacted by
email, included in the corresponding authors list, and will try their
best to provide additional guidance.

3  Methods
3.1  Elements
of Immunological

Synapses

Chapters 2–12 deal with methods to investigate particular subsystems that are likely to be applicable to any type of immunological
synapse. These include cytoskeleton, immunoreceptor microclusters, receptor trafficking in vesicles, cytoplasmic signaling complexes, and interfacial patterns. In some cases, the experimental
examples focus on Jurkat T cells, a common model system because
somatic variants lacking key signaling molecules are available and
they are readily transfectable to generate stable or transiently
expressing cell lines. But others provide examples with primary
cells. In one instance, the focus is on cell-free reconstitution of
signaling, which nicely complements in situ analysis of signaling
microclusters. This group also includes a chapter on mathematical
modeling of molecular patterns in the immunological synapse.

3.2  Technologies

Chapters 13–27 focus on technologies that can be applied to the
study of any immunological synapse. These include single molecule imaging and interaction measurements, fluorescence resonance energy transfer (FRET), force measurement, micro and


4

Michael L. Dustin and Cosima T. Baldari

nano-fabrication methods, proteomics, asymmetric cell division,
electron tomography, and systematic imaging methods. In some
instances these modalities are combined as in single molecule
FRET and force measurements using micro- or nano-fabricated
surfaces. Different approaches to systematic analysis of immune
synapses are highlighted. A powerful method for gene expression
in primary cells based on mRNA electroporation is described.

Finally, a chapter is provided on methods for recombinant protein
expression in bacterial or mammalian cells that provide greatly
accelerated pathways to milligram amounts of proteins, which are
enabling for many of the reconstitution approaches.
3.3  Biological
Examples

Chapters 28–36 focus on some compelling biological examples
studied using a variety of cutting edge methods. These include
analysis of killer cells in action, neuro-immune synapses, and consequences of pathological situations like cancer and infection for
immune synapses. The book ends with two protocols for in vivo
imaging of T cell-dendritic cell interactions in vivo, which is critical
for basic understanding and also to help guide the in vitro efforts
toward greater future relevance.
We are very excited to have these state-of-the-art methods,
most of which have already been featured in outstanding primary
publications, described in step by step detail in one volume. It is
our hope that this collection will accelerate the reproduction of key
results, prime new biological observations, and technical innovations. Best wishes for success with your experimental and/or modeling efforts.

Acknowledgments
We thank Éva Culleton-Oltay for assistance with editing the chapters and all the authors for their hard work on this project.
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12. Grakoui A, Bromley SK, Sumen C, Davis MM,
Shaw AS, Allen PM, Dustin ML (1999) The
immunological synapse: a molecular machine
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285(5425):221–227
13.Dustin ML, Olszowy MW, Holdorf AD, Li J,
Bromley S, Desai N, Widder P, Rosenberger F,
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A novel adapter protein orchestrates receptor
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14.Davis DM, Chiu I, Fassett M, Cohen GB,
Mandelboim O, Strominger JL (1999) The
human natural killer cell immune synapse. Proc
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15.Batista FD, Iber D, Neuberger MS (2001) B
cells acquire antigen from target cells after synapse formation. Nature 411(6836):489–494
16.Stinchcombe JC, Bossi G, Booth S, Griffiths
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17.Carroll-Portillo A, Spendier K, Pfeiffer J,
Griffiths G, Li H, Lidke KA, Oliver JM, Lidke
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Formation of a mast cell synapse: Fc epsilon RI
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Goodridge HS, Reyes CN, Becker CA,
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dendritic cell immunological synapse is

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J Leukoc
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(2002) Dynamic imaging of T cell-dendritic
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T cell priming by dendritic cells in intact lymph
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22.Azar GA, Lemaitre F, Robey EA, Bousso P
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107(8):3675–3680
23.Jacobelli J, Bennett FC, Pandurangi P, Tooley
AJ, Krummel MF (2009) Myosin-IIA and
ICAM-1 regulate the interchange between two
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Finetti F, Paccani SR, Riparbelli MG,
Giacomello E, Perinetti G, Pazour GJ,
Rosenbaum JL, Baldari CT (2009) Intraflagellar
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the TCR/CD3 complex to the immune synapse. Nat Cell Biol 11(11):1332–1339
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28.Schubert DA, Gordo S, Sabatino JJ Jr,
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17765–17770


Chapter 2
Analyzing Actin Dynamics at the Immunological Synapse
Katarzyna I. Jankowska and Janis K. Burkhardt
Abstract
T cell signaling is inextricably linked to actin cytoskeletal dynamics at the immunological synapse (IS). This

process can be imaged in living T cells expressing GFP actin or fluorescent F-actin binding proteins.
Because of its planar nature, the IS provides a unique opportunity to image events as they happen, monitoring changes in actin retrograde flow in T cells interacting with different stimulatory surfaces or after
pharmacological treatments. Here, we described the imaging methods and analytical procedures used to
measure actin velocity across the IS in T cells spreading on planar stimulatory surfaces.
Key words Actin, Cytoskeleton, Kymograph, Immunological synapse, T-cells, Integrin, Planar lipid
bilayer, Mobile ligands, Spinning disk, Live cell imaging

1  Introduction
The formation of the immunological synapse (IS) between a T cell
and an antigen presenting cell (APC) depends on actin dynamics
downstream of T cell receptor (TCR) and integrin engagement
[1–4]. TCR signaling activates the Arp2/3 complex-dependent
polymerization of branched actin filaments at the edges of the cell-­
cell contact site, driving initial spreading of the T cell on the APC
surface and subsequent centripetal flow of the acto-myosin network.
This process corresponds to the retrograde actin flow that occurs at
the leading edge of a migrating cell. Centripetal actin flow drives
the ongoing assembly and function of TCR signaling complexes,
and ultimately shuttles these complexes to the center of the IS,
where signal extinction takes place [5, 6]. Centripetal flow of the T
cell actin network also regulates integrin conformational change,
thereby promoting adhesion to ligands on the APC surface, as well
as outside-in signals that costimulate T cell activation [7, 8]. Actin
dynamics thus function as an essential part of a key feedback loop
that coordinates T cell signaling events at the IS. Thus, measuring
actin flow at the IS is valuable for u
­ nderstanding the fundamental
mechanisms that drive and fine-tune T cell activation.
Cosima T. Baldari and Michael L. Dustin (eds.), The Immune Synapse: Methods and Protocols, Methods in Molecular Biology,
vol. 1584, DOI 10.1007/978-1-4939-6881-7_2, © Springer Science+Business Media LLC 2017


7


8

Katarzyna I. Jankowska and Janis K. Burkhardt

Much of what is known about actin dynamics at the IS comes
from studies of T cells responding to coverslips or planar lipid
bilayers coated with stimulatory antibodies or ligands [9]. While
these planar stimulatory surfaces do not recapitulate the complex
undulations that are formed at a T cell-APC interface [10, 11],
they represent a powerful tool because they allow investigators to
image the movements of fluorescently tagged cytoskeletal elements
and signaling molecules in the microscope’s X–Y plane. In conjunction with these planar stimulatory surfaces, several labs have
taken advantage of super resolution approaches such as structured
illumination (SIM), stimulated emission depletion (STED), and
single molecule localization techniques (PALM/STORM) to
examine the molecular architecture at the IS [12–16]. Recently,
lattice light sheet technology has also proven to be valuable [17].
Nonetheless, simpler techniques for live cell imaging such as total
internal reflection (TIRF) and spinning disk confocal microscopy
continue to be the best ways to answer many biological questions.
TIRF optics are often used to image movement of signaling molecules at the IS. This modality is favored because it focuses analysis
on events occurring at or very near the plasma membrane (within
about 100 nm), and offers low background noise. However, the
actomyosin network extends more than 1 μm into the cell at early
times after TCR engagement [11]. Even at later times when T cells
are well spread, the actin-rich lamellipodia are nearly 0.5 μm thick

[18]. Thus, only a subset of actin filaments is captured within the
TIRF plane. Indeed, it is nearly impossible to gain an overall sense
of the acto-myosin network using TIRF optics. Because we are
interested in the actin cytoskeleton as a functional unit, we prefer
to use spinning disk confocal microscopy. As detailed below, we
typically capture three planes spanning a total distance of 0.5 μm.
This usually captures the entire thickness of the lamellipodium of a
spreading T cell. By generating a maximum intensity projection or
a 3-dimensional rendering of the three planes, we can analyze the
behavior of the lamellipodial actin network as a whole.
Armed with suitable video sequences, it is relatively straightforward to carry out quantitative analysis of actin flow rates by
tracking the movement of small structures within the actin network. The most common technique to visualize motion from
sequential 2-D imaging is kymographic analysis [19, 20]. To generate a kymograph, one first selects a narrow region of interest and
extracts this region from each image in a time series. The selected
region is then laid side-by-side for all time points, generating a
picture (kymograph) that displays movement of objects within the
selected region over time, such that one axis represents space and
the other axis represents time. Movement within these space-time
plots is seen as diagonal lines of bright or dark features, and the
speed of movement can be determined based upon the slope of
these lines.


Analyzing Actin Dynamics at the Immunological Synapse

9

In studies using planar surfaces to analyze protein dynamics at
the IS, one important factor is the mobility of stimulatory ligands.
Neither glass surfaces bearing immobilized ligands nor planar lipid

bilayers bearing freely mobile ligands faithfully recapitulate the
biology of bona fide antigen presenting cells, where some ligands
exhibit constrained mobility and others are freely mobile [21].
However, these simplified systems provide a means of exploring
the ways in which ligand mobility affects actin flow and signaling
through actin-coupled receptors.
There are several good protocols in the literature for the preparation of T cell stimulatory surfaces suitable for microscopy [22–
24]. Here, we describe our procedures for the preparation of both
immobile and mobile stimulatory surfaces, followed by our methods for imaging and measuring lamellipodial actin flow in T cells
interacting with these surfaces. We provide details for viewing actin
movements in a predetermined plane with optimal spatial and temporal resolution, and testing the effects of ligating specific receptors in the absence of other stimuli and altering actin dynamics
using pharmacological agents.

2  Materials
All solutions should be prepared using reverse osmosis (Milli-Q)
water and analytical grade reagents. Avoid repeated freeze-thawing
of protein solutions and inhibitors; make small aliquots before
freezing. Follow institutional safety guidelines in handling and disposing of hazardous chemicals including Hellmanex III and
Piranha solution and biohazardous materials such as recombinant
viruses and human cells.
2.1  Generation
of Stimulatory
Surfaces
2.1.1  Preparation
of Glass Coverslips

1.Coverslips for Sticky-Slides (Ibidi): # 1.5H (170 μm ± 5 μm)
D 263 M Schott glass, 25 mm × 75 mm.
2. 2% Hellmanex III detergent (Hellma Analytics): dissolve 20 ml
of detergent in 980 ml of water. Other alkaline glassware detergents may substitute for Hellmanex III (e.g., Linbro 7×), but

optimization of concentration, time, and rinsing requirements
is needed.
3.Piranha solution: Working in a fume hood, mix 300 ml of sulfuric acid with 100 ml of hydrogen peroxide in a glass beaker
[7] (see Note 1).
4.Bath sonicator.
5.Plasma cleaner.

2.1.2  Preparation
of Imaging Chambers

1.Bottomless Sticky-Slide VI 0.4 6-channel chambers (recommended supplier –Ibidi, Martinsried, Germany). Various home-­
made flow chambers may be substituted. The desired channel
width and length is ~5 × 25 mm with a height of 250 μm.


10

Katarzyna I. Jankowska and Janis K. Burkhardt

2.1.3  Coating Surfaces
with Immobilized Ligands

1.Mouse anti-human T cell receptor: OKT3 (recommended
supplier—BioXCell, Burlington, VT). Store at 4 °C.
2.Human VCAM-1: 1 mg/ml in PBS. Available as a soluble Fc
fusion from recommended suppliers Sino Biological or R&D
Systems. His-tagged protein also works for nonspecific adsorption. Store at −20 to −80 °C in small aliquots.
3.Human ICAM-1: 1 mg/ml in PBS. Available as a soluble Fc
fusion from recommended suppliers Sino Biological or R&D
Systems. His-tagged protein also works for nonspecific adsorption. Store at −20 to −80 °C in small aliquots.

4.L15 imaging medium: Supplement L15 with 2 mg/ml d-(+)
glucose by adding 1 g of d-glucose to 500 ml L15 medium
and filtering through a sterile filter unit (0.2 μm pore size).
Optionally, use phenol red-free RPMI-1640 enriched with 25
mM HEPES by adding 12.5 ml of sterile 1 M HEPES solution
to 500 ml RPMI medium.
5. Phosphate buffered saline (PBS)- standard formulation with or
without d-glucose.
6.Multichannel pipette.
7.Poly-l-Lysine hydrobromide (PLL) (mol. wt. 30,000–
70,000): Dissolve 5 mg in 10 ml of water. Add 40 μl of 5%
NaN3. Freeze in aliquots or store at 4 °C for 1–2 weeks.

2.1.4  Coating Surfaces
with Stimulatory Supported
Planar Lipid Bilayers

1.50 ml glass round-bottom flask.
2.Glass syringes (10, 50, and 500 μl, Hamilton).
3.2% Hellmanex III detergent: see Subheading 2.1.1, item 2.
4.Acetone (HPLC grade).
5.Chloroform (HPLC grade).
6. Lipids (recommended supplier—Avanti Polar Lipids, see Note 2):
DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) 25 mg/ml
in chloroform, DGS-NTA(Ni) (1,2-dioleoyl-sn-­
glycero-3[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl]
(nickel salt)) 10 mg/ml in chloroform, DSPE-­
PEG(2000)
Biotin (1,2-distearoyl-sn-glycero-3-­­phosphoethanolamine-N[biotinyl(polyethylene glycol)-2000] (ammonium salt) ) 5
mg/ml in chloroform. Store at −20 °C.

7.Extruder set (recommended supplier—Avanti Polar Lipids):
excluder with two glass syringes and 50 nm pore membranes.
8.Bath sonicator.
9.Compressed air (house air is fine, but place a filter on the output to remove contaminants).
10. Vacuum dessicator.
11. PBS.


Analyzing Actin Dynamics at the Immunological Synapse

11

12.Biotinylated OKT3 (0.5 mg/ml). Store at 4 °C.
13.Streptavidin, NeutrAvidin or NeutrAvidin –TexasRed conjugate: 1 mg/ml in PBS. Store at −20 °C.
14.Human VCAM-1-His tagged (recommended supplier—Sino
Biological Inc): 1 mg/ml in PBS. Store at −20 to −80 °C in
small aliquots.
15.Human ICAM-1-His tagged (recommended supplier—Sino
Biological): 1 mg/ml in PBS. Store at −20 to −80 °C in small
aliquots.
16. L15 imaging medium: L15 supplemented with 2 mg/ml d-(+)
glucose.
17. Multichannel pipette.
2.2  Imaging Actin
Dynamics in Living
T Cells
2.2.1  Preparation
of Cells Expressing
Fluorescent Actin Probes
for Live-­Cell Imaging


1.Jurkat T cell growth medium: RPMI 1640 enriched with 5%
fetal bovine serum and 5% newborn calf serum, 2 mM L-alanyl-­
l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin
(see Note 3).
2.Jurkat T cells stably expressing fluorescent actin probes (fluorescent protein-labeled actin, Lifeact, or F-tractin) (see Notes 3
and 4 for details on the use of these probes).

Jurkat T-Cells
Primary Human Peripheral
Blood CD4+ T Cells

1.Primary T cell growth media: RPMI 1640 supplemented with
10% fetal bovine serum (Atlanta Biologicals), 2 mM l-alanyl-­lglutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and
50 U/ml of human rIL-2 (see Note 5).
2.Primary human CD4+ T cells expressing fluorescent actin
probes (see Notes 3 and 6).

2.2.2  Fluorescence
Microscopy

Below is the description of the setup we use to acquire the images.
Other vendors also provide similar systems and there are many
options for analysis software.
1.Inverted microscope (Zeiss Axiovert 200 with Piezo Z-focus).
2.Yokagawa spinning disk head (PerkinElmer Ultraview ERS6
with Photokinesis unit).
3.CCD camera: Orca ER camera, Hamamatsu (see Note 7).
4.Objective: 63× Plan Apo 1.4 NA, oil immersion.
5.Solent Scientific environmental chamber.

6. Multi laser module (laser lines 405, 440, 488, 514, 561, 640 nm).
7.
Emission filters (455/60, 485/60, 527/55, 587/125,
615/70, 705/90).
8.Vibration isolation table (Vibraplane kinetic systems).
9.Image acquisition software (Volocity v. 6.3, Perkin Elmer).


12

Katarzyna I. Jankowska and Janis K. Burkhardt

2.3  Image Analysis

1.Volocity v. 6.3 imaging software (Perkin Elmer).
2.Microsoft Excel.

3  Methods
3.1  Generation
of Stimulatory
Surfaces
3.1.1  Preparation
of Glass Coverslips
(See Note 8)

1.Sonicate the glass coverslips in 2% Helmanex detergent for
30 min.
2.Wash vigorously with water to remove all detergent.
3.Air dry.
4.Dip into fresh Piranha solution and incubate for 30 min (see

Note 1).
5.Wash with water.
6.Air dry.
7.For studies involving lipid bilayers, we recommend plasma
cleaning the glass for 3 min just before the addition of the
small unilamellar liposome vesicle (SUV) solution. This step is
dispensable for studies where coverslips are coated with stimulatory proteins without lipid bilayers.

3.1.2  Preparation
of Imaging Chambers
(See Note 9)

1.Peel the protective paper off the Sticky-Slide 6-channel chambers, revealing the self-adhesive underside. Mount cleaned
coverslip, aligning carefully. Take care to touch only the edges
of the coverslip.
2. Press down carefully, using a pen or the back of a pair of tweezers to secure the seal between each well. To prevent leakage,
make sure that the tape sticks well to the coverslip. If desired,
one can further protect the sides from leaking by applying nail
polish around all sides.

3.1.3  Coating Surfaces
with Immobile or Mobile
Ligands

Two types of stimulatory surfaces can be prepared: ligand can be
immobilized by adsorption onto the glass coverslip (Subheading
“Coating with Immobile Ligands by Protein Adsorption to the
Coverslips”) or ligand can be attached to lipid bilayers where it will
have high lateral mobility (Subheading “Coating Surfaces with
Stimulatory Supported Planar Lipid Bilayers”).


Coating with Immobile
Ligands by Protein
Adsorption
to the Coverslips

We typically apply OKT3, a monoclonal antibody that reacts
with an epitope on the epsilon-subunit within the human CD3
complex [25], in the presence or absence of the adhesion molecules VCAM-1 or ICAM-1, which bind to the integrins VLA-4
and LFA-1, respectively. Depending on the experiment, other
stimulatory ligands can be used, and concentrations can be varied
(see Note 10).
1.Dilute OKT3 in PBS to a concentration of 10 μg/ml.


Analyzing Actin Dynamics at the Immunological Synapse

13

2. Using the multichannel pipette, add 100 μl of 10 μg/ml OKT3
to each chamber well. To create flow in one direction, it is
important always to pipette into one side of the wells (intake
ports), and out from the other side (outtake ports). To facilitate this, mark the intake side of the chamber (the side to which
OKT3 was added) with an arrow.
3.Incubate 2 h at RT or overnight at 4 °C.
4.Wash each well three times with PBS. To do this, use a multichannel pipette to remove 90% of the solution in the wells
(withdraw from the outtake ports). Then pipette in 150 μl of
PBS to the marked intake side. Repeat two more times. Never
let the wells dry out.
5. At this point, another ligand can be added. We use 2 μg/ml of

VCAM-1 or ICAM-1 in PBS. Withdraw 100 μl of PBS from
the outtake wells and replace with 100 μl of 2 μg/ml VCAM-1
or ICAM-1. Incubate for another 2 h at 37 °C, and wash three
times with PBS, all as in steps 2–5 (see Note 11).
6.Exchange the solution to L-15 imaging medium by washing
three times with pre-warmed L-15 medium (37 °C).
7.Incubate the chamber on the microscope stage at 37 °C for
about 10 min before adding cells.
Coating Surfaces with
Stimulatory Supported
Planar Lipid Bilayers

In order to facilitate specific binding of ligands to the lipid bilayer,
functionalized lipid must be incorporated into the lipid mixture
during vesicle preparation. Many functionalized lipids are commercially available. We used biotinylated lipids and lipids with a
Ni-NTA group, allowing us to attach biotinylated OKT3 (via a
Streptavidin bridge) as well as His-tagged ICAM-1 or VCAM-1.
The following procedure has two stages: Steps 1–13 describe
preparation of lipid vesicles in chloroform in a desired mol% ratio.
We use 5 mM DOPC:DSPE-PEG(2000) biotin:DGS-NTA(Ni)
(nickel salt) in a 98:1:1 mol% ratio. Steps 14–23 describe the use
of these vesicles to generate planar bilayers.
1.Sonicate the 50 ml glass round-bottom flask and the extruder
set in 1% Hellmanex III solution for 10 min.
2. Thoroughly rinse the flask and extruder set with water to completely remove the residual detergent.
3.Air dry.
4.Rinse the flask in acetone and then chloroform, vortexing to
be sure to cover all surfaces. It’s fine to leave a little chloroform
in the flask.
5.Wash each glass syringe thoroughly by passing chloroform

through it five to ten times.
6.Use the glass syringes to add 200 μl chloroform to the flask.
Again using the glass syringes, add 30.6 μl DOPC, 6 μl


14

Katarzyna I. Jankowska and Janis K. Burkhardt

DSPE-­PEG(2000), and 1 μl DGS-NTA into the flask, pipetting each into the chloroform. Vortex to mix (see Note 12).
7.Gently dry the lipid solution with compressed air while rotating the round bottle to make a uniform lipid film.
8.Place the round bottle into a vacuum desiccator and dry for 2
h or overnight.
9.Rehydrate the lipid film with 200 μl of PBS (or the buffer of
your choice) to bring the final lipid concentration to 5 mM,
and then sonicate for 5 min to produce micelles.
10.Assemble the mini-extruder as per Avanti instructions, using
the 50 nm pore membrane.
11.Pass PBS through the extruder a few times to ensure that the
assembly does not leak. Monitor the volume that comes across
the extruder after five to ten passes. If volume is lost, reassemble the set.
12.Extrude the lipid solution through the membrane at a constant, steady rate 21 times, creating a lipid vesicle mixture (see
Note 13).
13. Transfer the lipid vesicle mixture to a 1.5 ml conical microcentrifuge tube. This mixture can be kept at 4 °C for 1 week.
14. For each chamber slide, mix 150 μl of the lipid vesicle mixture
with 600 μl PBS to get 1 mM liposome suspension. Vortex to
mix (see Note 14).
15.Using freshly prepared plasma cleaned Sticky-Slide chambers
(see Subheading 3.1.2), and a multichannel pipette, add about
100 μl of diluted lipid vesicle mixture to each chamber and

incubate for 30 min at RT.
16. Rinse the wells thoroughly with PBS to remove the excess vesicles. This should be done by sequential addition of PBS to the
intake well and removal of flow-through on the other side.
About 150 μl of PBS can be added at a time for a total of three
to five times. Never allow the wells to dry out or air to enter
the channel containing the bilayer.
17. Remove the final PBS wash and pipette in Streptavidin or fluorescent Neutravidin (1 μg per 100 μl PBS for each well).
Incubate for 20 min at RT.
18.Wash thoroughly as before.
19.Incubate the chambers with biotinylated OKT3 and His-­
tagged ICAM-1 or VCAM-1. This should be done sequentially, incubating for 20–30 min and washing three times with
PBS after each addition (see Note 15).
20. Exchange the PBS with three washes of L-15 imaging medium.
21. Bilayer surfaces should be tested for ligand mobility (see Note 16)
and used the same day. We usually transfer them to the microscope


Analyzing Actin Dynamics at the Immunological Synapse

15

environmental chamber (see Subheading 3.2.2). Ligand mobility
and surface quality may decrease with longer storage.
22.Incubate the chamber on the microscope stage at 37 °C for
about 10 min before adding cells.
3.2  Imaging Actin
Dynamics in Living
T Cells

1.Culture T cells expressing fluorescent actin probes as detailed

in Notes 3–6. Ensure that the culture is growing well, and that
cells exhibit high viability at the time of analysis.

3.2.1  Preparation
of Cells Expressing
Fluorescent Actin Probes
for Live-­Cell Imaging
3.2.2  Preparation
of the Microscope

1.Set the environmental chamber on the microscope to 37 °C,
and allow it to equilibrate for at least 1 h prior to imaging.
2. Place all chambers, reagents, etc. into the environmental chamber to allow equilibration.

3.2.3  Preparation
of Cells for Live-­Cell
Imaging

1.Pipette about 5 ml of cells into a 15 ml tissue culture grade
conical tube.
2.Centrifuge the cell suspension at 250 × g for 5 min at room
temperature.
3.Aseptically aspirate or decant the supernatant without disturbing the cell pellet.
4.Resuspend the cell pellet in 5 ml of L-15 medium.
5.Determine the total number of cells using a hemocytometer.
6.Centrifuge the cells again to remove residual serum.
7.While the cells are in the centrifuge, calculate the volume of
L-15 imaging medium needed to resuspend the cells at the
desired density. We typically resuspend the cells at 1 × 106/ml.
8.Resuspend the washed cells in L-15 medium at the desired

final density. Maintain at 37 °C until imaging (see Note 17).

3.2.4  Imaging Cells
by Spinning Disk Confocal
Microscopy

1. Open imaging software and set all basic parameters. Configure
the time-lapse settings; we usually collect a z-stack of three
planes spaced 0.25 μm apart every 0.5–1 s, over a total time of
about 4 min.
2. Inject 50 μl of cell suspension into the intake well of a chamber
coated with stimulatory ligands (prepared as described in
Subheading 3.1).
3.Mix and distribute the cells by gently removing about 50 μl of
flow-through from the outtake port and adding back to the
intake port. Repeat this three to five times.