Methods in Molecular Biology 1186 Elena Ranieri Editor Cytotoxic T-Cells Methods and Protocols M M B ETHODS IN OLECULAR IOLOGY Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hat fi eld, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 Cytotoxic T-Cells Methods and Protocols Edited by Elena Ranieri Department of Medical and Surgical Sciences, University of Foggia, Foggia, Italy Editor Elena R anieri Department of Medical and Surgical Sciences University of Foggia Foggia, Italy ISSN 1064-3745 ISSN 1940-6029 (electronic) ISBN 978-1-4939-1157-8 ISBN 978-1-4939-1158-5 (eBook) DOI 10.1007/978-1-4939-1158-5 Springer New York Heidelberg Dordrecht London Library of Congress Control Number: 2014944379 © Springer Science+Business Media New York 2 014 This work is subject to copyright. 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Printed on acid-free paper Humana Press is a brand of Springer Springer is part of Springer Science+Business Media (www.springer.com) Prefa ce My memories trace back to when I was an 8-year-old girl, busy searching for information and illustrations about animals in natural science books; eager to search and continually learn. Books have been my world, and my thoughts and wishes have originated from them. Today, that girl has become a researcher who has edited the present textbook of molec- ular biology which is expected to stand as a p rimum movens that researchers may use while conducting a research activity applied to immunology; a textbook that can be used to start research for the characterization of Cytotoxic T Lymphocytes (CTL) by employing a sys- temic approach. The text is intended to outline basic and advanced laboratory methods in a way that will render straightforward and clear results. This book aims to be an introductory course to Systems Biology in Cytotoxic T Cells. The emerging area of Systems Biology is a holistic approach that strives for a system-level understanding of biology. This means assessing the complex dynamics of how the structure of cells and organisms functions together, rather than the characteristics of isolated parts of a cell or organism. One problem with this approach is that so far, no book has placed enough emphasis on laboratory methods applied to immunology. Our idea is to provide a book that introduces the concepts of Systems Biology along with the lab protocols that have generated such concepts. We stress the importance of offering clear explanations in order to show both students and researchers the relationship between experiments and concepts. The efforts and enthusiasm of all the contributing authors are apparent in this textbook that treats the main topic as an adventure in discovery. I personally thank all of them for taking part in this challenging project and for sharing their laboratory protocols. The text is written in a clear-cut manner and elegantly illustrated in full color. It com- prises 16 chapters. In the fi rst part the book focuses on the isolation of T cells (Chapter 1 ), their expansion and characterization according to different methods (Chapters 2 and 3 ) . The required techniques for intracellular signaling, monitoring of antigen T cell specifi c responses, and CTL exosomes are reported in Chapters 4 – 7 . Microscopy and in vivo imag- ing applied to CTL studies are found in Chapters 8 and 9 . “Omics” approaches are described in Chapters 10 – 13 . Finally, the last three chapters include a specialist application of molecu- lar methods into the study of CTL (Chapters 14 – 16 ) such as next generation sequencing of the Jack/stat pathway and CTL involvement in bone remodeling and transplantation. Foggia, Italy E lena Ranieri v Acknowledgement We thank the Section of Pathological Anatomy, Department of Emergency and Organ Transplantation, University of Bari “Aldo Moro,” Bari, Italy for kindly providing the cover image. vii Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Acknowledgement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi 1 N egative and Positive Separation Techniques for the Isolation of Antigen-Specific CD8+ T Cells from Blood and Tumor Tissue. . . . . . . . . . . 1 Margherita Gigante, S haron N atasha C ox, and Elena Ranieri 2 I n Vitro\Ex Vivo Generation of Cytotoxic T Lymphocytes . . . . . . . . . . . . . . . 1 3 Margherita Gigante and Elena Ranieri 3 P henotypic and Functional Characterization of Cytotoxic T Lymphocytes by Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Iulia P opescu, Matthew Pipeling, J ason A kulian, and John M cDyer 4 Intracellular Signaling of CTLs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 9 Sharon N atasha C ox 5 M onitoring Antigen-Specific T Cell Responses Using Real-T ime PCR. . . . . . . 6 5 Devin B . L owe, Jennifer L. Taylor, and W alter J. S torkus 6 C TL ELISPOT Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 5 Elena R anieri, I ulia P opescu, and M argherita G igante 7 Methods of Purification of CTL-Derived Exosomes . . . . . . . . . . . . . . . . . . . . 8 7 Angela M ontecalvo, Adriana T . Larregina, and A drian E. M orelli 8 C haracterization of CTL by Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 03 Giuseppe S tefano N etti 9 Intravital Imaging of Cytotoxic T Lymphocytes . . . . . . . . . . . . . . . . . . . . . . . 1 21 Gaetano Faleo, P er-Olof B erggren, and A ntonello Pileggi 10 T he Microarray-Based Approach for the Analysis of the Transcriptome . . . . . . 1 31 Matteo Accetturo, Paola Pontrelli, and Loreto Gesualdo 11 m iRNome Analysis Using Real-Time PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 01 Paola P ontrelli, M atteo A ccetturo, and Loreto G esualdo 12 Proteomic Approaches by SELDI and MALDI-TOF/MS for CTL Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233 Massimo Papale and Maria T eresa R occhetti 13 T wo-Dimensional Gel Electrophoresis Approach for CTL Phosphoproteome Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 43 Maria Teresa R occhetti, M assimo P apale, and L oreto Gesualdo 14 Targeting the JAK/STAT Pathway in Cytotoxic T lymphocytes (CTL) by Next Generation Sequencing (NGS) . . . . . . . . . . . . 2 53 Maddalena G igante, Sterpeta D iella, and E lena Ranieri ix