Methods in molecular biology vol 1584 the immune synapse methods and protocols

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 For further volumes: http://www.springer.com/series/7651 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 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 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   The Immune Synapse: Past, Present, and Future Michael L Dustin and Cosima T Baldari   Analyzing Actin Dynamics at the Immunological Synapse Katarzyna I Jankowska and Janis K Burkhardt   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   Analyzing the Dynamics of Signaling Microclusters Akiko Hashimoto-Tane, Tadashi Yokosuka, and Takashi Saito   Reconstitution of TCR Signaling Using Supported Lipid Bilayers Xiaolei Su, Jonathon A Ditlev, Michael K Rosen, and Ronald D Vale   Plasma Membrane Sheets for Studies of B Cell Antigen Internalization from Immune Synapses Carla R Nowosad and Pavel Tolar   Studying the Dynamics of TCR Internalization at the Immune Synapse Enrique Calleja, Balbino Alarcón, and Clara L Oeste   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   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 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 Studying Dendritic Cell-T Cell Interactions Under In Vivo Conditions 573 15 PBS 16 10 mM CellTracker Green (CMFDA, Thermo Fisher) in DMSO or other dye 17 10 mM CellTracker red (CMTPX, Thermo Fisher) in DMSO or other dye 18 Pluronic 19 Incomplete RPMI 20 31 G insulin needle 2.4  Popliteal Lymph Node Imaging Tools for dissection: scissors, forceps, microscissors (i.e., Vannas Spring Scissors—3 mm cutting edge, Fine Science Tools), ultrafine forceps (i.e., Dumont #5, Fine Science Tools) Isoflurane anesthesia system Surgical tape Dissecting microscope Heating pad Spray/squirt bottle containing 70% ethanol Electric hair clippers for shaving animals Depilatory cream for hair removal (i.e., Nair) Cotton buds 10 Imaging stage (see Fig 1) 11 Gauze pads 12 Phosphate buffered saline, PBS warmed to 37 °C 13 12 ml syringe for application of PBS 14 Vetbond glue 15 Glass coverslip 16 Vacuum grease 17 12 ml syringe 18 Two-photon laser scanning microscope fitted with epifluorescence illumination source, opaque environmental chamber, and high numerical aperture (NA = 1.0) water immersion objective (either ×20 or ×25 magnification) 19 NDD light path setup, as per manufacturer recommendation with 470/30 filter for CMF2HC photon collection, 525/50 for CMFDA photon collection, and 600/75 for CMTPX ­collection Optional, 400/75 filter for second harmonic collection on four channel microscopes 2.5  Image Analysis and Quantification of Interactions Software for analysis of data Commercial sources include MetaMorph (Molecular Devices), Imaris (Bitplane), and Volocity (PerkinElmer) Open-source software includes programs such as ImageJ, BioImageXD, Icy, and Fiji 574 Nicholas van Panhuys Fig Stage design for popliteal lymph node preparation Use 2 mm ferritic stainless steel for the base of the stage, non-stainless steel will rust rapidly due to the use of PBS, and ferritic steel will allow the attachments of small magnets for holding the lymph node preparation in place Secure 5 mm Perspex top, precut to the dimensions indicated using strong adhesive glue For bolts labeled (A), cut a small slot through their diameter to allow the insertion of a 23 G syringe needle (used to secure the thigh) For bolts labeled (B), use bolts with small washers to allow the secure attachment of the coverslip holder Coverslip holder should be made from aluminum to avoid altering the position of the magnets while imaging and to prevent rusting 3  Methods 3.1  Preparation of DCs for Transfer 3.1.1  Spleen Isolation Euthanize animals according to the ethical guidelines governing the use of animals in your laboratory (see Note 6) Place animal on dissection board in a supine position, and sterilize the mouse and dissection equipment with ethanol, before making a left paracostal incision of approximately 2 cm Locate the spleen and use forceps to gently remove it into a well of a six-well plate, containing 4 ml of digest media (cRPMI and 125 μl Liberase DL at 13.1 U/ml, final concentration 0.4 U/ml Up to five spleens can be digested per well) Carefully inject 3 ml of digest media into each spleen ensuring that the excess solution is collected back into the well containing the spleens Incubate spleens for 20 min in cell culture incubator Place spleens into a cell strainer atop a 50 ml tube, use 3 ml plunger to gently dissociate spleen tissue, and wash into 50 ml tube using cRPMI Centrifuge at 350 × g for 5 min Resuspend pellet in 400 μl MACS buffer and 90 μl CD11c beads per spleen Incubate for 15 min at °C (fridge) Add 20 times volume of MACS buffer and re-strain Studying Dendritic Cell-T Cell Interactions Under In Vivo Conditions 575 10 Wash via centrifugation at 350 × g for 5 min and proceed to MACS separation as per manufacturer’s instructions 11 To enhance CD11c cell purity, pass over a second MACS column 3.1.2  Antigen Loading and DC Stimulation Count cells, wash via centrifugation at 350 × g for min, and resuspend at × 106 cell/ml Plate out at ml/well in 24-well plates Add relevant adjuvant for DC activation and peptide for DC loading at desired concentrations (see Notes and 5) Incubate cells for h in cell culture incubators 3.1.3  Cell Staining and Adoptive Transfer of DCs Pool wells and count cells Wash cells in cold PBS via centrifugation at 350 × g for 5 min Prepare CellTracker Blue (CMF2HC) stain From 10 mM stock CMF2HC to a final concentration of 200 μM in PBS, add Pluronic at 1:10 of amount of CMF2HC used Use 1 ml stain per × 106 cells Incubate cells with CMF2HC in cell culture incubators for 20 min Wash cells in cRPMI × via centrifugation at 350 × g for 5 min Incubate cells in cRPMI in cell culture incubators for 20 min Wash in PBS via centrifugation at 350 × g for 5 min Count and wash cells in PBS via centrifugation at 350 × g for 5 min Resuspend cells at 1–2 × 106 per 25 μl in PBS Adoptively transfer CMF2HC stained CD11c+ DC into the right rear footpad of host animals using a 31 G insulin needle 3.2  Preparation of T Cells for Transfer 3.2.1  Tissue Isolation for TCR Tg and Control T Cells Euthanize animals according to the ethical guidelines governing the use of animals in your laboratory (see Notes 2, 3, and 6) Pin animal to dissection board in a supine position; sterilize the mouse and dissection equipment with ethanol before making requisite incisions required to locate the spleen, mesenteric, axial, and brachial lymph nodes; and use forceps to gently remove them into a well of a six-well plate, containing cRPMI Place lymph nodes and spleens into a cell strainer atop a 50 ml tube, use 3 ml plunger to gently dissociate tissues, and wash into the 50 ml tube using cRPMI Centrifuge at 350 × g for 5 min Lyse red blood cells in ACK solution or similar lysis solution For ACK usage resuspend cells in ACK for 5 min at room temperature 576 Nicholas van Panhuys Wash in MACS buffer by centrifugation at 350 × g for 5 min Resuspend pellet in 90 μl MACS buffer and 10 μl anti-CD4 or anti-CD8 beads per × 107 cells Incubate for 15 min at °C (fridge) Add 20 times volume of MACS buffer and re-strain Wash via centrifugation at 350 × g for 5 min and proceed to MACS separation as per manufacturer’s instructions 10 To enhance cell purity, pass over a second MACS column 3.2.2  Cell Staining and Adoptive Transfer of T Cells Wash cells in cold PBS via centrifugation at 350 × g for 5 min Prepare CellTracker Red (CMTPX) and CellTracker Green stain (CMFDA) from 10 mM stocks, CMTPX to a final concentration of 1.25 μM (1:8000) in PBS, and add Pluronic at 1:1 of amount of CMTPX used CMFDA to a final concentration of 1.0 μM (1:10,000) in PBS, add Pluronic at 1:1 of amount of CMFDA used Use 1 ml stain per × 106 cells Incubate TCR Tg T cells with CMTPX and control T cells with CMFDA in cell culture incubators for 15 min (or vice versa) Wash cells in incomplete RPMI × via centrifugation at 350 × g for 5 min Incubate cells in incomplete RPMI in cell culture incubators for 20 min Wash in PBS via centrifugation at 350 × g for 5 min Count and wash cells in PBS via centrifugation at 350 × g for 5 min Resuspend cells at × 106 per 50 μl in PBS Adoptively co-transfer × 106 TCR Tg T cells and × 106 control T cells into host animals 18 h post-transfer of DCs (see Note 7) 3.3  Preparation of Popliteal Lymph Node for Intravital Imaging Perform initial induction of anesthesia in a chamber at 2–4% isoflurane in 100% oxygen, and then secure anesthetic nose cone to the animal’s head with surgical tape and maintain anesthesia with 1–2% isoflurane in 100% oxygen Monitor respiration rates to maintain deep anesthesia Use electric hair clippers to shave the hair on the right hind leg of the animal (Fig 2a) Use cotton buds to apply depilatory cream to the shaved area for min, and thoroughly wash area with paper towels and water to remove hair and excess cream (Fig 2b) Transfer animal to imaging stage in a prone position, and secure nose cone to the stage Use surgical tape and Vetbond Studying Dendritic Cell-T Cell Interactions Under In Vivo Conditions 577 Fig Surgical preparation of animal for imaging (a–g) As described in Subheading 3.3 Preparation of Popliteal Lymph Node for Intravital Imaging to secure the right footpad to the base of the imaging stage Use surgical tape to secure the left leg and tail to the left side of the stage (Fig 2c and d) Use a 23 G needle to secure the leg to the stage via holes in bolts (refer to bolts labeled A in Fig 1), and place the small magnets on either side of the leg to stabilize the tissue surrounding the popliteal lymph node (Fig 2e) Place the animal and stage onto a warming pad located under a dissecting microscope Sterilize the skin with ethanol and make a small incision in the middle of the back of the right thigh (Fig 2f) Locate the lymph node in the popliteal fossa by using the ultrafine forceps to carefully separate the covering tissue layer by layer Place a layer of gauze on top of the magnets on each side of the leg and moisten using PBS. Gauze pads should be kept moistened during imaging to prevent tissue dehydration and to help maintain stability of the LN preparation (Fig 2g) Fill 12 ml syringe barrel with vacuum grease, and apply a thin layer along all four edges of a coverslip, attach to coverslip holder, and slot onto the stage Moisten the area surrounding 578 Nicholas van Panhuys the LN and lower the coverslip so that it is in contact with the LN Avoid applying too much pressure, as this will inhibit cell migration (Fig 2h and i) 3.4  Acquisition of Two-Photon Intravital Images Pre-warm the environmental chamber on the two-photon microscope to 37 °C Add H2O to the top of the reservoir on the coverslip created with the vacuum grease Using the epifluorescent light source, locate and focus the water-dipping lens on the LN Tune the laser to the appropriate wavelength (Fig 3) to excite the fluorophores used to stain the cells, and locate area of interest to be imaged Adjust laser intensity to the minimum power required to illuminate the cells of interest in order to avoid phototoxicity (see Note 8) Fig Experimental design for multicolor two-photon imaging (a) Normalized emission intensities for the dyes outlined in the experimental procedures dependent on the excitation wavelength of the two-photon laser used In order to maximize the collection of photons from all three dyes, a two-photon excitation wavelength of 770–800 nm should be used However, it should be noted that as the excitation wavelength decreases, the background may increase, thus the optimal wavelength should be determined for each preparation (b) Comparison of the emission wavelength spectra of the dyes used in the experimental procedure Dyes are selected to minimize overlap in spectral emissions (c) Excitation and emission maximal values for common dyes and fluorescent proteins used in two-photon imaging Studying Dendritic Cell-T Cell Interactions Under In Vivo Conditions 579 Set imaging parameters: Z-stack approximately 40–80 μm with a distance between Z-layers of or μm Time between Z-stacks should be set to 15–45 s, and the time interval should be minimized by adjusting the scan speed and size of Z-stack in order to attain data allowing accurate tracking of individual cells Adjust digital zoom to an appropriate level to maximize number of interactions vs degree of detail (×1.0–2.0) Acquire images During imaging the animal’s breathing rate, gauze pads, and reservoir should all be checked every 30 min to ensure proper anesthesia is maintained, LN is kept moist, and the reservoir does not dry out Subsequent to the experimental endpoint, euthanize animals according to the ethical guidelines governing the use of animals in your laboratory 3.5  Analysis of Intravital Images and Quantification of Interactions Analysis of interaction lengths between DCs and T cells can be measured either by direct or indirect methods Direct methods involve either manual measurement of the length of interactions or automated methods [14, 15], which rely on measuring interactions by assessing the colocalization of voxels from cells with alternately colored stains For these automated methods, contact times between cells need to be specifically validated for each individual data set being quantified Direct manual measurement: control and TCR Tg cells tracked using software-driven algorithms DC present in imaged volume should be mapped and numbered for future reference (Fig 4b) To determine DC interaction history, each DC should be assessed on a per cell basis to determine whether a specific T cell is in contact with the DC during each of the time points imaged (Fig 4c) To determine T cell interaction history, each T cell should be assessed on a per cell basis to determine whether a specific DC is in contact with the T cell during each of the time points imaged, by following the previously calculated migratory path of the cell Indirect methods of measuring alterations in cellular interaction dynamics between populations can be applied by analyzing the migratory paths of cells [16] as determined in 3.2.2 –– Cellular velocity: when T cells interact with DC, their cellular velocity tends to decrease to