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

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

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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: http://www.springer.com/series/7651 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   Introduction to Homeostatic Migration Mark C Coles   Analysis of Thymocyte Migration, Cellular Interactions, and Activation by Multiphoton Fluorescence Microscopy of Live Thymic Slices Jessica N Lancaster and Lauren I.R Ehrlich   Visualizing and Tracking T Cell Motility In Vivo Robert A Benson, James M Brewer, and Paul Garside   Graph Theory-Based Analysis of the Lymph Node Fibroblastic Reticular Cell Network Mario Novkovic, Lucas Onder, Gennady Bocharov, and Burkhard Ludewig   Visualizing Endogenous Effector T Cell Egress from the Lymph Nodes Manisha Menon, Alexandre P Benechet, and Kamal M Khanna   Introduction: T Cell Trafficking in Inflammation and Immunity Myriam Chimen, Bonita H.R Apta, and Helen M Mcgettrick   Leukocyte Adhesion Under Hemodynamic Flow Conditions Charlotte Lawson, Marlene Rose, and Sabine Wolf   Endocrine Regulation of Lymphocyte Trafficking In Vitro Bonita H.R Apta, Myriam Chimen, and Helen M Mcgettrick   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 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 238 Robert A. Benson et al 1 ml Syringe and 27-G needle to inject the TCR Tg cells intravenously Warming cabinet and mouse restrainer for intravenous injection of isolated TCR transgenic CD4 T cells 2.3  Imaging Setup CS-12R coverslips, Harvard Apparatus High-vacuum silicone grease, BDH from VWR Various intubation cannulas with Luer adaptor, Harvard Apparatus: 1 mm OD 28 mm length; 1.1 mm OD 28 mm length; 1.2 mm OD, 30 mm length Standard isoflurane unit and isoflurane Standard preparation of mouse anesthetic, e.g., ketamine and xylazine Mini-vent and power supply, Hugo Sachs Elektronik, Harvard Apparatus Vacuum unit, Dymax 5, Charles Austin Imaging window unit, custom made by Almond Engineering, see Fig. 1a for specification 1 mm and 10 mm diameter tubing 10 Heat mat and homeothermic monitor, Harvard Apparatus 11 Plastic board with imaging window unit mount The mount consists of a series of optical posts and angle clamps (ThorLabs) providing an adjustable but rigid arm to which the imaging window is mounted See Fig. 1b 12 Surgical thread 13 Sharp dissecting tools including Castro-Viejo scissors 14 Aluminum foil 15 Cotton swabs and 70% ethanol to sterilize the skin 16 A multiphoton microscope, e.g., Zeiss LSM 7MP equipped with a 20×/1.0NA water-immersion objective lens 17 Appropriate analysis software, e.g., Volocity, PerkinElmer or Imaris, Bitplane 3  Methods 3.1  Infection of Mice with IAV Prepare influenza virus in 20 μl of sterile PBS (the virus dose depends on viral stock, strain and origin or mice, and type of experiment, see Note 1) Anesthetize mice with isoflurane in a sealed box; see Note Once the breathing of the mouse is slow and steady, remove the animal from the anesthesia box, and hold it gently in the T Cell Recruitment in Infection 2.10 A 239 12.60 13 7 5.10 M1.40 08 18 90 SCALE 5:1 1.60 SCALE 5:1 B Arm to mount imaging window Stand to secure imaging window arm Imaging window Tubing to attach to vacuum Board to arrange mouse on (along the black line) Fig A customized imaging window unit was made by Almond Engineering (a) It is made up of a stable arm connected to a small circular unit that contains a hole in which a round coverslip is inserted The imaging window unit is connected to a vacuum via tubing The unit is secured to a standard chopping board, shortened to fit on the microscope stage, with a customiszed holder (Thorlabs) (b) palm of one hand with the mouse in the supine position with its head elevated Slowly pipette the diluted virus in 20 μl onto the external nares Allow the mouse to regain consciousness before returning to its cage 240 Robert A. Benson et al 3.2  Analysis of IAV-Specific CD4 and CD8 T Cells by Flow Cytometry Euthanize mice either with a rising concentration of carbon dioxide or cervical dislocation; see Note The peak of the T cell response to IAV in the lung is between days and 12 post-­ infection; memory T cells can reliably be found up to 60 days after infection If required to remove blood cells from the lung, perfuse the mouse Euthanize the mouse using carbon dioxide if you plan to this To perfuse the mouse, open up the chest cavity after death has been confirmed, snip the aorta below the heart, and slowly inject 5–10 ml of room-temperature PBS with mM EDTA into the left ventricle The lungs should become pale white Snip out the lungs, carefully avoiding the thymus and the mediastinal lymph node, and place them in a tube of RPMI on ice prior to processing If desired, take the mediastinal lymph node, other lymph nodes, and spleen for analysis of IAV-specific T cells in lymphoid organs These can be processed into a single-cell suspension either using a 70 μm 50 ml tube strainer or between two pieces of gauze or two slides To digest the lungs, first place them in a dry Petri dish and cut into small pieces using scissors Transfer the lung pieces into a tube and add 1 ml of RPMI Once all the lungs have been processed, add 1 ml of the 2× digestion mix Alternatively stagger the addition of the digestion mix if many samples are being processed Incubate the lungs in the digestion mix at 37 °C for 40 min If you have access to a shacking incubator, shake slowly If not, incubate in a water bath and mix the samples every 10–20 min After the incubation, gently shake the tube to resuspend the lung pieces and pour the contents onto a 70 μm tube strainer placed into a 50 ml Falcon tube With a plunger from a 2.5 ml syringe, macerate the lung pieces into the cell strainer Rinse the strainer with 5 ml of RPMI, and continue mashing until the lung pieces have disintegrated Rinse with a further 5 ml of RPMI 10 Pellet the cells at 400 × g for 5 min at 4–8 °C 11 If mice were not perfused, or perfusion was incomplete, lyse red blood cells using red blood cell lysis buffer, use ml, and incubate at room temperature for 1–2 min 12 Wash cells twice with 5–10 ml of RPMI 13 Count cells using a hemocytometer, and expect to see around 20–40% cell death T Cell Recruitment in Infection 241 14 During the primary response (days 8–15), the sample can be split into 5–8 samples; at memory time points (after days 20–30) 2–4 samples as the percentage of IAV-specific T cells will be much reduced 15 To stain cells with MHC tetramers: transfer around 200– 500,000 cells in 100–200 μl into a 96-well round-bottom plate Include wells for control unstained, single-stained samples and “fluorescence-minus-one” samples as appropriate We usually use spleen or lymph node cells for this purpose 16 Prepare diluted tetramer in tetramer staining media 17 Spin plate at 400 × g for 3–5 min, discard supernatant, and blot the plate on absorbent paper to remove all traces off the supernatant 18 Add the diluted tetramer in 25–40 μl, gently pipetting each sample up and down to ensure that the cells are not clumped 19 Wrap the plate in cling film/saran wrap and place at 37 °C 20 Stain for 1–2 h, with gentle agitation of the plate every 20–30 min For these reasons we leave empty rows and columns between samples on the plate to remove any risk of contamination by spillage between wells 21 Surface antibodies can be added directly at the correct pre-­ dilution to the cells or following a wash Stain for 10–20 min at room temperature or °C 22 Wash cells either with PBS if preceding to viability stain or with FACS buffer if not 23 Prepare viability dyes in PBS. Stain for 20 min at °C and then wash twice with FACS buffer 24 Stained cells can be fixed in 1–4% PFA for convenience, for intracellular staining, or if cells were taken from mice with live virus Fix for 20–30 min at °C or room temperature 25 Cells can be acquired on a standard flow cytometer as per the manufacturer’s instructions and analyzed using standard analysis software such as FlowJo 26 A typical gating analyses to identify IAV-specific CD4 and CD8 cells is shown in Fig. 2 3.3  Transfer of Fluorescent TCR Tg Cells for Intravital Imaging Harvest lymph nodes and spleens from OT-II animals [12] crossed to mice that intrinsically express a fluorescent protein, e.g., CD2-DSRed transgenic animals [11] Isolate CD4 T cells, e.g., using magnetic beads; we use negative selection beads from either Miltenyi Biotec or Stemcell Technologies following the manufacturer’s instruction It is good practice to confirm successful isolation of CD4+ T cells by flow cytometry 242 Robert A. Benson et al Fig Example gating for IAV-specific CD4 and CD8 T cells Lung cells from a naïve C57BL/6 mouse or a mouse infected intranasally with WSN 30 days earlier were prepared as described and stained with APC-Db/ NP366-74 tetramer and PE-IAb/NP311–325 tetramer, anti-CD4 Alexa-Fluor 700, anti-CD44 APC-Alexa780, anti-CD8 Pe-Cy7, anti-TCRβ PerCP-Cy5.5, anti-B220-eFluor 450, anti-MHC II-eFluor 450, anti-F4/80 eFluor 450, and fixable viability dye eFluor 506 Cells were acquired on an LSR and analyzed using FlowJo software Cells were sequentially gated through a lymphocyte gate, a single-cell gate, a gate for live cells before gating on TCR+ “dump” (eFluor 450) negative cells that were then split into CD4+ or CD8+ cells Finally, MHC tetramer+ cells were identified as tetramer+ CD44+ cells The numbers show the percentages of cells in the indicated gates Remove all protein (FCS) from the buffer the cells are in by washing twice in PBS or HBSS and transfer five million CD4 T cells intravenously into host mice We use mice that express EYFP under the control of the CD11c promoter [10] which in the lung identifies dendritic cells and alveolar macrophages [13] The following day, infect the mice as above (see Subheading 3.1) with influenza virus that expresses the OT-II epitope, ovalbumin 323–339 [12] T cells can typically be found in the lung from day post-­ infection onwards; we have not examined time points after day 11 of infection 3.4  Intravital Imaging This procedure was developed by Krummel and colleagues and is described in some detail in Ref Please refer to this publication for images of the mouse surgery T Cell Recruitment in Infection 243 Fig Overview of lung imaging setup The anesthetized mouse is positioned on a heat mat secured to the imaging board (1) The imaging board has a stand for the imaging window unit (2) which is connected to the vacuum (3) An oxygen unit (4) drives isoflurane (5), through a mini-vent unit, (6) which controls the breathing of the mouse following intubation Tubing connects the mini-vent to the mouse through a cannula (7) The ventilator exhaust is placed in a beaker with about 2 cm of water (8) A scavenger (9) is connected during imaging but only switched on following imaging to prevent excessive outflow pressure on the lungs It is good practice to run through the procedure on euthanized animals prior to using live mice An overview of the imaging setup is shown in Fig. 3 Ensure that microscope system is fully operational prior to collecting sample tissue: use test slide check laser and detectors Ensure that appropriate filters and mirror combinations are in place for the fluorophores in use 3.5  Preparation of the Imaging Window Unit Ensure that the unit and tubing are clean and dry Secure the imaging window unit to the window arm which should be attached to the mouse board, Fig. 1b Position a dissecting microscope over the imaging window unit With a clean pair of forceps, pick up a round coverslip and check that it fits perfectly into the inner ring of the imaging window unit 244 Robert A. Benson et al Suck some vacuum grease up into a 2.5 ml syringe Squirt a thin line of vacuum grease onto some absorbent paper and gently roll the outer rim of the cover slip in the grease ensuring that the grease does not come into contact with the center of the coverslip Carefully place the coverslip into the inner ring of the imaging window and secure by running the forceps around the edge of the coverslip 3.6  Preparation of the Vacuum Unit Cut off and discard the narrow end of a 1 ml filter pipette tip Attach the wider end (containing the filter) to a piece of 10 mm tubing and the narrower end to a piece of 1 mm tubing to connect the two pieces of tubing The tubing should be long enough to comfortably stretch from the vacuum unit to the surgery area and the microscope stage The filter ensures that any blood which enters the tubing does not enter the vacuum Connect the vacuum tubing up to the vacuum unit at one end and the imaging window unit at the other end 3.7  Preparation of the Mouse Surgery Board Place the board on the microscope stage and mark out where the mouse will need to be positioned to ensure that the lungs and imaging window will lie under the objective lens Ensure that the stage is still able to move freely Wrap the small heat mat up in aluminum foil and secure it to the center of the mouse board with tape Turn on the homeothermic mat and allow it to reach 37 °C. Secure a temperature probe inside the homeothermic mat to monitor the temperature during imaging Cut two pieces of surgical thread approximately 2 cm long and put to one side Cut 5–10 pieces of tape approximately 5 cm long; stick to the side of the bench for easy access 3.8  Preparation of the Mini-Vent Connect the tubing up: the mini-vent should be connected to the isoflurane unit via the inflow and the tubing that connects to the mouse cannula via the outflow The ventilator exhaust should be placed in a beaker with about 2 cm of water This produces a positive-end expiratory pressure, avoiding damage to the lungs Set the mini-vent: typically ventilate at 100 μl/10 g of mouse weight, 200 μl for a 20 g mouse The breathing rate is governed by the number of strokes per minute which should be approximately 130–150 Connect the outflow tube to an intubation cannula of appropriate size for the mouse; it should fit snugly into the trachea T Cell Recruitment in Infection 3.9  Mouse Surgery 245 Anesthetize the mouse with an injectable anesthetic Check that the animal is fully anesthetized by testing for a response after pinching between its toes Place the mouse on the heat mat on its back With a cotton swab gently rub a small amount of ethanol on the mouse’s skin under its chin Cut a square of about 1 cm2 area of skin below the mouse’s chin being careful not to cut any blood vessels Gently prise away the tissue below the skin pulling the tissue apart from either side to reveal the trachea Using curved forceps, pull the surgical thread under the trachea Make a small hole in the trachea using Castro-Viejo scissors Quickly insert the cannula into the hole, and gently push it deeper into the trachea Quickly connect the breathing apparatus up to mini-vent and turn it on The mouse’s breathing is now controlled by the mini-vent: check that it is steady 10 Quickly turn on the isoflurane unit at a low level, about 2% Do not turn on the scavenger during imaging as this causes excessive outflow pressure on the lung Do make sure that the outflow is connected to the scavenger to draw in the isoflurane Turn the scavenger on at the end of imaging to remove any remaining isoflurane 11 Secure the cannula in the trachea by tying two tight knots in each of the pieces of surgical thread Secure the cannula and tubing to the board with tape 12 Make sure that the bottom of the mouse’s lungs is aligned with the black line on the board 13 With the mouse on its back, tape down the hind limbs, with the left leg loosely held at first 14 Place a 1 ml pipette tip diagonally under the mouse to push the rib cage upwards 15 Tape the fore limbs: the right front limb is taped down followed by the left front limb which is pulled up and over the mouse’s head so that the mouse is now lying on its right side Adjust the tape on the left hind limb to be more secure 16 Wipe the area around the mouse’s left rib cage with ethanol Using Castro-Viejo scissors gently remove the skin around this area to expose the lung 17 Gently, with two pairs of forceps, peel the top layer of tissue away 18 Pick up the third and fourth ribs from the bottom of the rib cage with two pairs of forceps Firmly but carefully pull these apart to tear a hole in the rib cage Be very careful not to touch 246 Robert A. Benson et al the lungs; this will cause bleeding and lung imaging will be very difficult or impossible At this stage the lungs should drop because of the difference in pressure, but the mouse will continue breathing because of the ventilator 19 Using the Castro-Viejo scissors, snip out and remove the ribs An area larger than the imaging window must be exposed 20 Turn the vacuum on, and place the imaging window unit gently down onto the surface of the lung The vacuum should cause the imaging window unit to seal to the lungs, but the imaging window unit should not be pressing down on the lungs 21 Carefully move the setup to the microscope stage ensuring that no tubes are pulled out and that no objects are placed on top of any tubes 22 Focus using the objective before recording images and movies, e.g., Fig. 4 Please refer to Benson et al in this issue for more details refer Chap 23 At the end of the imaging session, euthanize the animal humanly (do not allow to recover from anesthesia), e.g., by cervical dislocation, transfer to a CO2 chamber, or injection of euthatal (150 mg/kg i.v.) 24 Use software analysis, e.g., Volocity, to analyze T cell displacement, velocity, meandering index, and contact time with antigen-­presenting cells Fig Example intravital lung images CD11c-EYFP mice adoptively transferred with × 106 CD4 T cells isolated from OT-II X CD2-DSRed mice, were infected intranasally with WSN-OVA 11 days before intravital imaging was performed Red OT-II cells can be found in close proximity to green CD11c+ cells in some cases Blue signal represents second harmonics from collagen White arrows point to OT-II cells; b is a zoomed-in image of the white boxed area in a, a tile scan of the lung analyzed using Volocity T Cell Recruitment in Infection 247 4  Notes We typically use WSN at 300PFU per mouse and PR8 at 50PFU per mouse These doses cause between 10 and 20% weight loss in C57BL/6 mice that are between 10 and 16 weeks of age Mice that lose greater than 20% of their starting weight must be euthanized for health reasons With these doses, we typically have to euthanize 10% of infected animals For imaging ­experiments, we drop the dose slightly so the animal is not overly stressed prior to imaging Mice recover from isoflurane within minutes, so they must be carefully monitored Slower regular breathing is a good indication of anesthesia (if shallow and fast, the mouse is not properly anesthetized); mice should not exhibit response to pain (test via gently squeezing the footpad) The mouse must not be left unattended throughout the duration of this procedure In some instances, it may be desirable to inject intravenously a fluorescently labeled antibody into the mice 3–5 min prior to euthanasia This will label cells in the circulation, leaving cells in the tissue unlabeled In this case, it is not necessary to perfuse the animals to remove blood cells from the lungs This procedure has been described in depth [14] Acknowledgments  We are grateful to Prof Matthew Krummel, University of California, San Francisco, and his laboratory for sharing the methodology of the intravital lung imaging technique and to Prof James Brewer and Dr Ross McQueenie for their assistance in setting up this method at Glasgow University We acknowledge the NIH Tetramer Core Facility (contract HHSN272201300006C) for provision of MHC tetramers Dr MacLeod is an Arthritis Research UK Fellow (Grant ID: 19905) References Guilliams M, Lambrecht BN, Hammad H (2013) Division of labor between lung dendritic cells and macrophages in the defense against pulmonary infections Mucosal Immunol 6(3):464–473 doi:10.1038/mi.2013.14 Duan S, Thomas PG (2016) Balancing immune protection and immune pathology by CD8(+) T-cell responses to influenza infection Front Immunol 7:25 doi:10.3389/fimmu 2016.00025 Lim K, Hyun YM, Lambert-Emo K, Capece T, Bae S, Miller R, Topham DJ, Kim M (2015) Neutrophil trails guide influenza-specific CD8(+) T cells in the airways Science 349(6252):aaa4352 doi:10.1126/science aaa4352 248 Robert A. Benson et al Brown DM, Lampe AT, Workman AM (2016) The differentiation and protective function of cytolytic CD4 T cells in influenza infection Front Immunol 7:93 doi:10.3389/ fimmu.2016.00093 Strutt TM, McKinstry KK, Marshall NB, Vong AM, Dutton RW, Swain SL (2013) Multipronged CD4(+) T-cell effector and memory responses cooperate to provide potent immunity against respiratory 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9(1):209–222 doi:10.1038/nprot.2014.005 Index A Adherent cells��������������������������� 102, 109, 110, 112–114, 118, 128, 132, 134, 135, 137, 138, 157, 164, 188, 226 Adhesion����������������������������������������������������� 75–78, 80, 99, 156, 170–173, 178, 191, 216 cell adhesion molecules α4β7�������������������������������������������������������������� 171, 172 ICAM-1����������������������������������������������� 75, 77, 78, 80 immunoglobulin super family����������������������� 191, 216 integrins����������������������������������������������������������������171 JAMs��������������������������������������������������������������������171 L-selectin��������������������������������������������������������������216 selectins�����������������������������������������������������������������170 VAP-1�������������������������������������������������������������������178 VCAM-1��������������������������������������75–78, 80, 99, 156, 170, 171, 173, 178, 216 multistep cascade����������������������������������������������������75–77 Adoptive transfer���������������������������������������������� 28–29, 36, 60 Agarose tissue blocks����������������������������������������������������������17 Albumin�������������������������������������������103, 114, 122, 145, 159, 178, 180, 184, 191, 192, 227 Anesthesia���������������������������������������31, 36, 39, 238, 246, 247 Antibody����������� 44–46, 61–63, 69, 95, 146, 147, 150, 152, 172, 180, 185, 188, 198, 200, 211, 222, 235, 237, 247 Antigen-driven T cell migration���������������������������������������217 Antigen presenting cells (APCs)����������������������� 2, 10–12, 27, 28, 86, 90, 96, 246 Arterial vessel�������������������������������������������������������������������206 Arteries������������������������������������������������������������� 127, 128, 206 Artery����������������������������������������������������������������������� 173, 195 B Biotinylation��������������������������������������������� 187–189, 217, 219 Bone marrow�����������������������������������������������3, 28, 32, 33, 169 Bovine serum albumin (BSA)�������������������������� 103, 105, 108, 110, 111, 115, 122, 131, 133, 134, 139, 145, 146, 150, 159, 163, 164, 218, 221, 227, 228 C Calcium flux�����������������������������������������������������������������11, 23 Calcium levels���������������������������������������������������������������������11 Ccr7−/−��������������������������������������������������������������������������������11 CD3ε������������������������������������������������������������������������ 228, 234 CD4��������������������������3, 12, 14, 29, 32–33, 38, 87, 88, 90, 91, 96–98, 117, 140, 166, 185–186, 200–201, 205, 206, 211, 218, 220, 228, 234–238, 240–242, 246 CD4+������������������������������������������� 94, 181, 186, 191, 198, 199 CD45��������������������������������������������������������������� 228, 233, 234 CD4−CD8���������������������������������������������������������������������������3 CD8��������������������������������������������������� 12, 14, 60, 63, 67, 117, 140, 166, 235–237, 240–242 CD8+��������������������������������������������������������������������������������178 CD19����������������������������������������������������������������������� 228, 234 CD31���������������77, 89, 98, 171, 172, 217, 219, 223, 228, 233 Cdc42��������������������������������������������������������������� 145, 146, 153 CD326 (EpCAM)���������������������������������������������������� 228, 233 Cell cultures�������������������������������� 13, 18, 19, 90–91, 103, 105, 116, 124, 139, 148, 158–159, 180, 191, 198, 201, 217, 218, 227 Cell tracking���������������������������� 11, 28, 70, 186–187, 211, 221 CellTrackerTM�������������������������������������������������������������������182 CellTracker fluorescent dyes CMFDA��������������������������������������������������������� 29, 33, 39, 182, 186, 199 CMPTX�����������������������������������������������������������������28, 32 CFSE������������������������������������������������89, 91, 95–97, 218, 221 Chronic inflammation������������������������������������������������ 78, 169 CMRA��������������������������������������������������������������������� 218, 221 Coculture���������������������������������� 74, 79, 93–98, 122, 129–136, 140, 178, 201, 202, 205, 211 Collagen gels�������������������������������������������80, 122–124, 130–136, 140, 141, 203, 205 Collagenase collagenase D�������������������������������������� 203, 226–233, 237 collagenase P������������������������������������������������������227–233 Cre�������������������������������������������������������������������� 44, 45, 47, 63 Cryosections������������������������������������������������������������� 189, 192 Cytokines��������������������������������������� 2, 4, 5, 43–47, 59, 60, 78, 80, 101, 102, 115, 171–173, 216 adipokines adiponectin��������������������������������������������������� 102, 115 leptin��������������������������������������������������������������� 78, 101 chemokines CCL19���������������������������� 2, 4, 5, 43–47, 80, 171, 216 CCR5��������������������������������������4, 59, 60, 80, 172, 216 CCR9����������������������������������������������������������� 172, 173 Cytoskeleton���������������������������������������������������������������������171 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, © Springer Science+Business Media LLC 2017 249 T-Cell Trafficking: Methods and Protocols 250  Index    H D Davidson Marking System dyes������������������� 199, 207, 209 3D reconstructions����������������������������������������6, 38, 44, 45, 47 Dendritic cell (DC)������������������� 2–4, 12, 28, 43, 78, 235, 242 Deoxyribonuclease I (DNAse I)������������������������������� 227, 228 Drug development������������������������������������������������������������170 TM E Egress��������������������������������������������������������4, 5, 59, 65–67, 69 Endocrine�����������������������������������������������������������������101–118 Endogenous T cells���������������������������������������������������� 63, 236 Endothelial cell expansion������������������������������������������������139 Endothelial cell freezing����������������������������� 97, 103, 107, 116 Endothelial cell isolation���������������������������� 98, 114, 126–129 Endothelial cells (EC) blood vascular endothelial cells��������������� 73, 80, 121–141 HDBEC������������������������������103, 105–107, 109, 113–116 HUVEC����������������� 87–99, 102, 105, 114, 115, 124, 127, 130, 132, 133, 135, 138–141, 156–158, 161–167 liver sinusoidal endothelial cells (LSEC)���������������200, 203 lymphatic endothelial cells (LEC)��������������������� 4, 60, 68, 73, 77, 80 murine endothelial cells������������������������������ 217, 219–220 Ethylenediaminetetraacetic acid (EDTA)����������������88–90, 103, 104, 106, 122, 123, 126, 129, 146, 159–161, 198, 199, 201, 204, 217–220, 227–229, 231, 236, 240 F Fetal calf serum (FCS)�����������������������������28, 29, 33, 88, 103, 123, 180, 199, 227–229, 231 Fibroblastic reticular cells (FRC)���������������2, 4, 43, 47, 50–52 Fibroblasts�������������������������������� 2, 43, 47, 50–52, 79, 80, 102, 121, 123, 126–129, 139, 140, 226 Firm adhesion�������������������������������74, 77, 136, 143, 170, 171 Flow cytometry���������������������������������������������32, 91, 94–98, 117, 140, 162, 163, 166, 199, 202, 204, 205, 211, 218, 222–223, 227, 229–233, 236, 237, 240–241 Flow rates������������������������������� 34, 92, 97, 102, 111, 113, 117, 137, 138, 141, 209, 213 Flow-based adhesion assay���������������������������������������124–126 Fluorescent reporter mice CD11c-YFP���������������������������������������������������������������237 hCD2-DsRed������������������������������������������������������� 29, 237 Foetal calf serum (FCS)��������������������116, 123, 129, 130, 139, 140, 145, 148, 185, 186, 209, 217–220, 237, 242 FTY720�������������������������������������������������������������� 4, 60, 62, 68 G Glutathione-S-transferase (GST)������������ 145, 146, 148–151 gp38 (Gylcoprotein 38/Podoplanin)������������������������� 228, 233 Granzyme B (GzmB)��������������������������������������� 60, 63, 65, 67 Graph theory������������������������������������������������������������ 6, 43–56 GTPase��������������������������������������������������������������������� 144, 152 Hepatic epithelia��������������������������������������������������������������199 Hepatocytes���������������������������������������� 178, 179, 185, 191, 196, 199, 200, 203, 205 Histo-morphometric analysis���������������������������������������������45 Hoeschst dye������������������������������������������������������������ 180, 185 Human��������������� 1, 10, 78, 80, 86–88, 90, 96–98, 102–105, 123, 126, 146, 156, 158, 162, 163, 169, 170, 172–174, 177–181, 185–186, 189, 195–213, 218 Human liver�������������������������������������178, 179, 182, 184, 186, 195–207, 209, 211, 213 I Ileum������������������������������������������������������������������������ 230, 232 Image analysis methods������������������������������������������������������������ 21–23, 40 software�������������������������������������� 13, 21, 32, 37, 104, 110, 112, 125, 134, 138, 159 Imaging window������������������������������������������������������ 238, 239, 243–244, 246 Imaris����������������������������������13, 21, 22, 32, 37, 38, 45, 47–49, 51, 52, 61, 62, 64–67, 69, 238 Immune system���������������������������������������1, 78, 102, 169, 235 Immunohistochemistry������������������������������ 44–46, 89, 94–95 In situ tetramer staining����������������������������������� 60, 68, 70, 71 In vitro������������������������������������ 5, 74, 80, 81, 86, 87, 101–118, 155–157, 162, 170, 179, 195–213, 217, 222 In vivo������������5, 27–40, 44, 63, 74, 78, 80, 81, 86, 87, 102, 155, 156, 166, 170, 174, 203, 217, 218, 221, 223, 236 Indo1AM����������������������������������������������������12, 15, 20, 22, 24 Infections�������������������������������� 5, 6, 59–61, 63, 67, 68, 73, 79, 116, 157, 179, 191, 215, 235–247 Inflammation��������������������������������������5, 73–81, 85, 101, 102, 155, 169–171, 173, 179, 196, 197, 215 Influenza A virus (IAV)�������������������������������������������235–242 Integrins���������������������������������������������9, 76–78, 80, 116, 143, 171–173, 191, 192, 196, 197, 216, 235 Intracellular cytokine staining���������������������������������� 202, 205 J Joints��������������������������������������������������������������������������������173 K Krumdieck tissue slicer��������������179, 180, 182–184, 199, 206 L Lasers����������������������������������13, 18, 20, 21, 24, 27, 28, 32, 33, 35, 37, 45–47, 61, 62, 64, 243 Lentiviral transduction��������������������������������������������� 157, 165 Leucotaxis������������������������������������������������������������������������170 Leukocyte������������������������������������������������������������� 59–71, 178 B cells������������������������������������������������2, 87, 102, 216, 235 lymphocyte�������������������������������������������������������������73–75 T-Cell Trafficking: Methods and Protocols 251 Index       peripheral blood lymphocytes (PBL)����������������� 102–105, 108–118, 126, 131, 133–135, 140, 148, 156, 159, 162–164, 166, 167, 185, 188 T cells CD8+ T cells���������������������������������������������������������178 effector T cells��������������������������������������������������59–71 Leukocyte recruitment��������������������74, 75, 78, 115, 121, 173 Leukocytes�����������������������������������������������������������������������156 Lineage depletion���������������������������������������������������������12, 14 Liver��������������������������������������������14, 173, 177–192, 195–213 Liver sinusoidal endothelial cells (LSEC)���������������� 200, 203 Liver wedges����������������������������������������������������� 199, 206–211 Lungs��������������������������������������������������14, 217, 219, 235–247 Lymph node (LN)����������������������������1, 2, 4–6, 27, 29–38, 43–47, 50–52, 59, 60, 65–67, 69, 74, 80, 169, 216, 220–223, 225, 227, 229–233, 235, 240, 241 Lymphatics�������������2, 4, 5, 36, 60, 62–68, 70, 73, 79, 80, 215 Lymphocyte isolation��������������������������������������� 114, 126, 159 M Mechanical digestion���������������������������������������� 186, 206, 211 Medical tissue marking dye����������������������������������������������209 Memory�������������������������������������� 1, 76, 77, 86, 117, 140, 156, 166, 169, 236, 240, 241 Microchannel������������������������������������������������������������� 74, 111 Mesenchymal stem cells (MSC)�������������79, 121, 123, 126–129 MHC tetramers migration������������������������������������ 62, 67–69, 236–237, 241, 242 Microscopy confocal microscopy�������������������������28, 44–47, 69, 74, 95 fluorescence microscopy����������������������������������� 13, 20, 97, 122, 124, 126, 132, 159 intravital imaging����������������������������10, 28, 60, 61, 64–67, 70, 241–243, 246 intravital microscopy����������������60, 61, 63–67, 74, 80, 174 multiphoton microscopy������������������������������� 5, 27, 32, 61 phase contrast microscopy����������������������94, 97, 105, 106, 109, 122, 124, 136, 145, 159 photon microscopy���������������������������������������� 10, 67, 74 Migration migration velocities����������������������109, 114, 117, 135, 139 MTT���������������������������������������������������������������� 180, 184, 185 N Network analysis���������������������������������������� 45, 47, 49–51, 53 O Organ cultures����������������������������������������������������������177–192 Oxygen concentrator���������������������������������������������� 29, 31, 34 P Parallel-plate flow chamber��������� 87–89, 91–98, 115, 124, 126 Peptide inhibitor of trans-endothelial migration (PEPITEM)��������������������������������������������� 78, 102 Perfusion chamber������������������������������������������������������������������19, 34 gas���������������������������������������������������������������������������11, 19 heater���������������������������������������������������������������� 13, 19, 34 medium������������������������������������������������������������ 13, 19, 23 Peristaltic pump����������� 29, 34, 91–93, 199, 206, 208–209, 213 Peritoneal lavage���������������������������������������������������������������221 Phenotype������������������������������������������ 49, 75, 87, 98, 99, 121, 177, 178, 186, 189, 192, 199, 202, 206, 236 Precision cut liver slices (PCLS)��������������� 179, 199, 205–206 Pull-down assay��������������������������������������������������������143–153 R Rac1������������������������������������������������������������������������� 145, 146 Recruitment������������ 74–80, 101, 102, 110, 115, 121–141, 156, 170, 173, 177–179, 185–189, 216, 218, 221–223 Resident immune cells������������������������������ 185, 186, 191, 192 RhoA���������������������������������������������������������������� 144–146, 148 RhoGTPase��������������������������������������������������������������143–153 Rolling cells�������������������������������������������������������������� 112, 138 Roswell park memorial institute (RPMI)�������������� 12, 13, 15, 16, 18, 19, 28, 29, 33, 62, 68, 88, 123, 127, 145, 150, 181, 186–188, 198, 199, 201, 202, 208, 209, 217, 218, 227–229, 231, 237, 240 S Salivary glands������������������������������������������ 225, 227, 229–233 SDS-polyacrylamide gel electrophoresis (SDS-PAGE)����������������������������������������� 147, 151 Secondary lymphoid tissue����������������������������������� 3, 4, 27, 28 Separated flows�������������������������������������������������������������97–98 Shear stress���������86–87, 91, 93, 105, 111, 117, 126, 137, 141 Short-hairpin-loop RNAs (shRNA)��������������������������������156 Sjögren’s syndrome����������������������������������������������������� 79, 225 Skin����������������������14, 31, 35, 36, 64, 123, 128, 159, 238, 245 Specimen harp��������������������������������������������������������������13, 20 Sphingosine phosphate (S1P)�����������������4, 5, 60, 68, 78–80 Sphingosine phosphate receptor (S1PR1)���������������� 59, 60, 63, 65, 68, 79 Spinbar® magnetic stirring fleas����������������������������������������227 Spleen���������������������������������������������������3, 186, 195, 220–223, 225, 227, 240, 241 Splenocytes��������������������������������������������������������������� 220, 222 Stamper Woodruff adhesion assays�������������������������� 182, 188 Static adhesion assay���������� 109, 112, 131, 156, 159, 162, 166 Stromal cells������������������������������� 2, 4, 6, 9–11, 20, 22, 43, 74, 75, 79–81, 102, 121–141, 174, 225 Systems biology������������������������������������������������������������������48 T T cell proliferation��������������������������������������������������������������97 Tamoxifen��������������������������������������������������������������� 60, 61, 63 Tansmigration������������������������������������������������������������������109 TCR activation������������������������������������������������� 10, 11, 22, 23 T-Cell Trafficking: Methods and Protocols 252  Index    TCR transgenic OT-II���������������������������������� 29, 32, 38, 39, 237, 241, 246 Tertiary lymphoid tissue������������������������������������������� 225, 227 Thermocouple��������������������������������������������������������������13, 16 Thymic slices������������������������������������������������������������������������9 Thymocyte����������������������������������������������������������������� 3, 9–24 Thymus����������������������������������� 3–6, 9–11, 14, 16, 23, 24, 240 Tissue digestion��������������������������������������������������������225–234 Tissue dye������������������������������������������206, 207, 209, 210, 212 Topological robustness�������������������������������������������������44, 50 Topology�������������������������������������������������6, 44, 45, 47, 50, 51 Trafficking������������������������������ 43, 59, 73, 101–118, 169–174, 177–192, 216, 226 Trans-gel assay������������������������������������������ 198–199, 203–205 Transmigraion�������������������������� 74, 76, 78, 87, 102, 108–110, 112–114, 117, 118, 131–133, 135, 138, 140, 141, 143, 164, 166, 167, 170–172, 199, 203, 205, 215 Transwell assay�����������������������������������������132, 141, 197–203, 205, 211, 220, 222 Transwell filters����������������������������������������������������������������141 V Vascular vessels�����������������������������������������������������������������206 Veins����������������������������������������� 33, 36, 39, 87, 102, 104, 105, 126–128, 156, 158, 170, 195, 197, 206 Velocity��������������������������������������� 6, 22, 38, 76, 109, 112, 114, 117, 134, 135, 137, 138, 171, 172, 246 Viability���������������������������������������� 95, 96, 115, 139, 166, 180, 184–185, 223, 237, 241, 242 W Western blotting�������������������������������������������������������� 94, 147, 151–153, 162 ... 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, ... 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. .. 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

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Mục lục

  • Preface

  • Contents

  • Contributors

  • Chapter 1: Introduction to Homeostatic Migration

    • 1 Introduction

    • 2 Migration in Immune Cell Development

    • 3 Migration in Immune Cell Function: Entry to Exit

    • 4 Imaging Immune Responses: Multiphoton Microscopy

    • 5 3-Dimensional Tissue Imaging

    • 6 Quantifying Biology and the Emergence of Modeling Technologies

    • 7 Conclusions

    • References

    • Chapter 2: Analysis of Thymocyte Migration, Cellular Interactions, and Activation by Multiphoton Fluorescence Microscopy of Live Thymic Slices

      • 1 Introduction

      • 2 Materials

        • 2.1 Preparation of Thymocytes

        • 2.2 Generation of Thymic Slices

        • 2.3 Seeding of Labeled Thymocytes into Thymic Slices

        • 2.4 Acquisition by Multiphoton Microscopy

        • 2.5 Image Analysis of Slices

        • 3 Methods

          • 3.1 Preparation of Thymocytes

            • 3.1.1 Harvest Thymocytes

            • 3.1.2 Enrich the Desired Thymocyte Subset

            • 3.1.3 Label Thymocytes with a Fluorescent Probe

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