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      Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)

      research-article
      1 , 2 , 27 , 2 , 3 , 4 , 2 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 7 , 9 , 20 , 21 , 22 , 23 , 24 , 25 , 2 , 6 , 26 , 28 , 29 , 14 , 30 , 18 , 31 , 32 , 33 , 34 , 22 , 35 , 28 , 2 , 8 , 36 , 1 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 2 , 49 , 50 , 28 , 24 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 9 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 37 , 65 , 66 , 9 , 19 , 3 , 4 , 28 , 29 , 1 , 51 , 67 , 68 , 28 , 29 , 3 , 69 , 70 , 71 , 72 , 73 , 74 , 32 , 33 , 42 , 76 , 77 , 78 , 79 , 80 , 45 , 46 , 21 , 22 , 63 , 81 , 82 , 83 , 13 , 84 , 85 , 7 , 86 , 87 , 21 , 22 , 88 , 2 , 89 , 90 , 66 , 65 , 91 , 79 , 80 , 28 , 29 , 92 , 83 , 93 , 94 , 52 , 94 , 65 , 51 , 95 , 2 , 21 , 22 , 96 , 2 , 8 , 97 , 37 , 22 , 96 , 98 , 8 , 99 , 100 , 83 , 101 , 2 , 8 , 28 , 2 , 2 , 102 , 88 , 42 , 103 , 104 , 105 , 79 , 80 , 42 , 62 , 63 , 41 , 106 , 12 , 51 , 107 , 108 , 2 , 82 , 101 , 3 , 24 , 99 , 50 , 61 , 15 , 109 , 59 , 60 , 88 , 17 , 49 , 75 , 33 , 3 , 103 , 110 , 111 , 24 , 112 , 66 , 77 , 78 , 10 , 113 , 95 , 30 , 53 , 54 , 17 , 79 , 80 , 10 , 93 , 6 , 114 , 6 , 115 , 79 , 80 , 88 , 91 , 2 , 88 , 2 , 41 , 79 , 80 , 34 , 116 , 117 , 24 , 77 , 78 , 48 , 2 , 51 , 107 , 118 , 119 , 120 , 2 , 32 , 33 , 37 , 5 , 117 , 3 , 94 , 36 , 21 , 22 , 89 , 90 , 61 , 121 , 122 , 48 , 2 , 17 , 113 , 123 , 88 , 32 , 33 , 82
      European journal of immunology

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          Abstract

          The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers.

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          Hallmarks of Cancer: The Next Generation

          The hallmarks of cancer comprise six biological capabilities acquired during the multistep development of human tumors. The hallmarks constitute an organizing principle for rationalizing the complexities of neoplastic disease. They include sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis. Underlying these hallmarks are genome instability, which generates the genetic diversity that expedites their acquisition, and inflammation, which fosters multiple hallmark functions. Conceptual progress in the last decade has added two emerging hallmarks of potential generality to this list-reprogramming of energy metabolism and evading immune destruction. In addition to cancer cells, tumors exhibit another dimension of complexity: they contain a repertoire of recruited, ostensibly normal cells that contribute to the acquisition of hallmark traits by creating the "tumor microenvironment." Recognition of the widespread applicability of these concepts will increasingly affect the development of new means to treat human cancer. Copyright © 2011 Elsevier Inc. All rights reserved.
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            Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

            ABSTRACT The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.
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              Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.

              The considerable therapeutic potential of human multipotent mesenchymal stromal cells (MSC) has generated markedly increasing interest in a wide variety of biomedical disciplines. However, investigators report studies of MSC using different methods of isolation and expansion, and different approaches to characterizing the cells. Thus it is increasingly difficult to compare and contrast study outcomes, which hinders progress in the field. To begin to address this issue, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy proposes minimal criteria to define human MSC. First, MSC must be plastic-adherent when maintained in standard culture conditions. Second, MSC must express CD105, CD73 and CD90, and lack expression of CD45, CD34, CD14 or CD11b, CD79alpha or CD19 and HLA-DR surface molecules. Third, MSC must differentiate to osteoblasts, adipocytes and chondroblasts in vitro. While these criteria will probably require modification as new knowledge unfolds, we believe this minimal set of standard criteria will foster a more uniform characterization of MSC and facilitate the exchange of data among investigators.
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                Author and article information

                Journal
                1273201
                3613
                Eur J Immunol
                Eur J Immunol
                European journal of immunology
                0014-2980
                1521-4141
                1 May 2024
                December 2021
                07 December 2021
                23 May 2024
                : 51
                : 12
                : 2708-3145
                Affiliations
                [1 ]Department of Medical and Surgical Sciences for Children & Adults, University of Modena and Reggio Emilia, Modena, Italy
                [2 ]German Rheumatism Research Center Berlin (DRFZ), Berlin, Germany
                [3 ]Istituto Nazionale di Genetica Molecolare Romeo ed Enrica Invernizzi (INGM), Milan, Italy
                [4 ]Department of Clinical Sciences and Community Health, Università degli Studi di Milano, Milan, Italy
                [5 ]Swiss Institute of Allergy and Asthma Research (SIAF), University of Zurich, Davos, Switzerland
                [6 ]Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, Munich, Germany
                [7 ]Division of Immunology, Transplantation and Infectious Diseases, IRCSS San Raffaele Scientific Institute, Milan, Italy
                [8 ]Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy
                [9 ]Mucosal Immunology Lab, Unidad de Excelencia Instituto de Biomedicina y Genética Molecular de Valladolid (IBGM, Universidad de Valladolid-CSIC), Valladolid, Spain
                [10 ]Institute of Immunology, Christian-Albrechts Universität zu Kiel & Universitätsklinik Schleswig-Holstein, Kiel, Germany
                [11 ]Institute of Clinical Molecular Biology Christian-Albrechts Universität zu Kiel, Kiel, Germany
                [12 ]Division of Medical Sciences, National Cancer Centre Singapore, Singapore
                [13 ]Cancer & Stem Cell Biology, Duke-NUS Medical School, Singapore, Singapore
                [14 ]Dipartimento di Medicina Interna e Specialità Mediche, Sapienza Università di Roma, Rome, Italy
                [15 ]Center for Life Nano & Neuro Science@Sapienza, Istituto Italiano di Tecnologia (IIT), Rome, Italy
                [16 ]Istituto Pasteur - Fondazione Cenci Bolognetti, Rome, Italy
                [17 ]Institute of Immunology, Hannover Medical School, Hannover, Germany
                [18 ]Medical Clinic III for Oncology, Hematology, Immuno-Oncology and Rheumatology, University Hospital Bonn, University of Bonn, Bonn, Germany
                [19 ]Laboratory of Molecular Genetics, Servicio de Análisis Clínicos, Hospital Universitario Río Hortega, Gerencia Regional de Salud de Castilla y León (SACYL), Valladolid, Spain
                [20 ]Centro de Investigaciones Biomédicas en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Madrid, Spain
                [21 ]Department of Surgery, The University of British Columbia, Vancouver, Canada
                [22 ]BC Children’s Hospital Research Institute, Vancouver, Canada
                [23 ]Department of Immunology and Pathology, Monash University, Melbourne, Victoria, Australia
                [24 ]Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin, Germany
                [25 ]Department of Microbiology & Immunology, Columbia University, New York City, USA
                [26 ]German Center for Infection Research (DZIF), Munich, Germany
                [27 ]Institute for Biotechnology, Technische Universität, Berlin, Germany
                [28 ]Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia
                [29 ]Australian Research Council Centre of Excellence in Advanced Molecular Imaging, University of Melbourne, Parkville, Victoria, Australia
                [30 ]Institute for Research in Biomedicine, Università della Svizzera italiana, Bellinzona, Switzerland
                [31 ]Instituto de Estudios Inmunológicos y Fisiopatológicos - IIFP (UNLP-CONICET), Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
                [32 ]Peter Gorer Department of Immunobiology, School of Immunology and Microbial Sciences, King’s College London, UK
                [33 ]National Institute for Health Research (NIHR) Biomedical Research Center (BRC), Guy’s and St Thomas’ NHS Foundation Trust and King’s College London, London, UK
                [34 ]Department of Viral Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany
                [35 ]Department of Medicine, The University of British Columbia, Vancouver, Canada
                [36 ]Infection and Immunity Program, Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia
                [37 ]Laboratory of Translational Immunology, IRCCS Humanitas Research Hospital, Rozzano, Milan, Italy
                [38 ]IRCCS, Istituto Giannina Gaslini, Genova, Italy
                [39 ]Institute for Immunology, University Medical Center Mainz, Mainz, Germany
                [40 ]Research Centre for Immunotherapy, University Medical Center Mainz, Mainz, Germany
                [41 ]Institute of Molecular Biology and Pathology, National Research Council of Italy (CNR), Rome, Italy
                [42 ]Immunosurveillance Laboratory, The Francis Crick Institute, London, UK
                [43 ]Innate Immunity Unit, Department of Immunology, Institut Pasteur, Paris, France
                [44 ]Inserm U1223, Paris, France
                [45 ]Laboratory of Innate Immunity, Department of Microbiology, Infectious Diseases and Immunology, Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany
                [46 ]Mucosal and Developmental Immunology, German Rheumatism Research Center Berlin (DRFZ), Berlin, Germany
                [47 ]Cell Biology, German Rheumatism Research Center Berlin (DRFZ), An Institute of the Leibniz Association, Berlin, Germany
                [48 ]Department of Medicine/Rheumatology and Clinical Immunology, Charité Universitätsmedizin Berlin, Berlin, Germany
                [49 ]Institute of Systems Immunology, Hamburg Center for Translational Immunology (HCTI), University Medical Center Hamburg-Eppendorf, Hamburg, Germany
                [50 ]Institut National de la Sante Et de la Recherce Medicale (INSERM) U1015, Equipe Labellisee-Ligue Nationale contre le Cancer, Villejuif, France
                [51 ]Singapore Immunology Network (SIgN), Agency for Science, Technology and Research, Singapore, Singapore
                [52 ]Institute of Transplant Immunology, Hannover Medical School, Hannover, Germany
                [53 ]Regensburg Center for Interventional Immunology (RCI), Regensburg, Germany
                [54 ]Chair for Immunology, University Regensburg, Regensburg, Germany
                [55 ]Institut Necker Enfants Malades, INSERM U1151-CNRS, UMR8253, Paris, France
                [56 ]Université de Paris, Paris Descartes, Faculté de Médecine, Paris, France
                [57 ]AP-HP, Hôpital Necker Enfants Malades, Paris, France
                [58 ]Department of Medicine I, Lighthouse Core Facility, Medical Center – University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
                [59 ]John van Geest Cancer Research Centre, School of Science and Technology, Nottingham Trent University, Nottingham, UK
                [60 ]Centre for Health, Ageing and Understanding Disease (CHAUD), School of Science and Technology, Nottingham Trent University, Nottingham, UK
                [61 ]Immunology of Aging, Leibniz Institute on Aging – Fritz Lipmann Institute, Jena, Germany
                [62 ]Department of Medicine, Visceral and Thoracic Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
                [63 ]Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
                [64 ]Hamburg Center for Translational Immunology (HCTI), University Medical Center Hamburg-Eppendorf, Germany
                [65 ]Division of Molecular Oncology and Immunology, the Netherlands Cancer Institute, Amsterdam, The Netherlands
                [66 ]Institute of Molecular Medicine and Experimental Immunology, Faculty of Medicine, University of Bonn, Germany
                [67 ]Shanghai Institute of Immunology, Department of Immunology and Microbiology, Shanghai Jiao Tong University School of Medicine, Shanghai, China
                [68 ]Translational Immunology Institute, SingHealth Duke-NUS Academic Medical Centre, Singapore, Singapore
                [69 ]Institute of Experimental Immunology, University of Zurich, Zurich, Switzerland
                [70 ]Department of Hematology, Oncology, and Tumor Immunology, Charité - Universitätsmedizin Berlin (CVK), Berlin, Germany
                [71 ]Berlin Institute of Health (BIH), Berlin, Germany
                [72 ]German Cancer Consortium (DKTK), partner site Berlin, Germany
                [73 ]Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, Victoria, Australia
                [74 ]Department of Molecular and Translational Sciences, Monash University, Clayton, Victoria, Australia
                [75 ]Peter Gorer Department of Immunobiology, King’s College London, London, UK
                [76 ]Division of Allergy, Department of Dermatology and Allergy, University Hospital Schleswig-Holstein, Kiel, Germany
                [77 ]Innate Immunity, German Rheumatism Research Center Berlin (DRFZ), Berlin, Germany
                [78 ]Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Department of Gastroenterology, Infectious Diseases, Rheumatology, Berlin, Germany
                [79 ]Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Department of Medicine 3 – Rheumatology and Immunology and Universitätsklinikum Erlangen, Erlangen, Germany
                [80 ]Deutsches Zentrum für Immuntherapie, Friedrich-Alexander-University Erlangen-Nürnberg and Universitätsklinikum Erlangen, Erlangen, Germany
                [81 ]Institute for Immunology, Biomedical Center, Faculty of Medicine, LMU Munich, Planegg-Martinsried, Germany
                [82 ]Experimental Immunology, Helmholtz Centre for Infection Research, Braunschweig, Germany
                [83 ]Department of Hematology, Oncology and Rheumatology, University Heidelberg, Heidelberg, Germany
                [84 ]Department of Hematology, Singapore General Hospital, Singapore, Singapore
                [85 ]Executive Offices, National Cancer Centre Singapore, Singapore
                [86 ]Vita-Salute San Raffaele University, Milan, Italy
                [87 ]Experimental Imaging Center, IRCCS San Raffaele Scientific Institute, Milan, Italy
                [88 ]Division of Molecular Immunology, Nikolaus-Fiebiger-Center, Department of Internal Medicine III, University of Erlangen-Nürnberg, Erlangen, Germany
                [89 ]Department of Rheumatology and Clinical Immunology, Medical Center – University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
                [90 ]Center for Chronic Immunodeficiency, Medical Center – University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
                [91 ]Institute of Molecular and Clinical Immunology, Otto-von-Guericke University, Magdeburg, Germany
                [92 ]Department of Neuropathology, Universitätsklinikum Erlangen, Germany
                [93 ]Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands
                [94 ]Institute for Molecular Medicine, Goethe University Frankfurt, Frankfurt am Main, Germany
                [95 ]Institute of Microbiology, ETH Zurich, Zurich, Switzerland
                [96 ]School of Biomedical Engineering, The University of British Columbia, Vancouver, Canada
                [97 ]Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham, UK
                [98 ]Michael Smith Laboratories, The University of British Columbia, Vancouver, Canada
                [99 ]Division of Infection & Immunity, Institute of Immunity & Transplantation, University College London, London, UK
                [100 ]Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA
                [101 ]Institute for Systemic Inflammation Research, University of Luebeck, Luebeck, Germany
                [102 ]Clinical Trial Center Leipzig, Leipzig University, Härtelstr.16, −18, Leipzig, 04107, Germany
                [103 ]Department of Immunology, IRCCS Bambino Gesù Children’s Hospital, Rome, Italy
                [104 ]Radiation Immuno-Oncology Group, Center for Translational Cancer Research (TranslaTUM), Technical University of Munich (TUM), Klinikum rechts der Isar, Munich, Germany
                [105 ]Department of Radiation Oncology, Technical University of Munich (TUM), Klinikum rechts der Isar, Munich, Germany
                [106 ]Institute of Experimental Immunology and Hepatology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
                [107 ]Department of Microbiology & Immunology, Immunology Programme, Life Science Institute, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
                [108 ]School of Biological Sciences, Nanyang Technological University, Singapore, Singapore
                [109 ]Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy
                [110 ]School of Biomedical and Health Sciences, RMIT University, Bundorra, Victoria, Australia
                [111 ]Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia
                [112 ]Flow Cytometry Science Technology Platform, The Francis Crick Institute, London, UK
                [113 ]Immunology Frontier Research Center, Osaka University, Japan
                [114 ]Clinic for Orthopedics and Trauma Surgery, University Hospital Bonn, Bonn, Germany
                [115 ]Mikrobiologisches Institut – Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität (FAU) Erlangen-Nürnberg, Germany
                [116 ]Charité Universitätsmedizin Berlin – BIH Center for Regenerative Therapies, Berlin, Germany
                [117 ]Sanquin Research – Adaptive Immunity, Amsterdam, The Netherlands
                [118 ]Institute for Medical Informatics, Statistics and Epidemiology (IMISE), University of Leipzig, Leipzig, Germany
                [119 ]Department of Precision Instrument, Tsinghua University, Beijing, China
                [120 ]Department of Preclinical Development and Validation, Fraunhofer Institute for Cell Therapy and Immunology IZI, Leipzig, Germany
                [121 ]Institute of Biochemistry and Biophysics, Faculty of Biological Sciences, Friedrich-Schiller-University Jena, Jena, Germany
                [122 ]Department of Medicine III, Technical University Dresden, Dresden, Germany
                [123 ]Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA
                Author notes
                Correspondence: Prof. Andrea Cossarizza and Dr. Hyun-Dong Chang, andrea.cossarizza@ 123456unimore.it ; chang@ 123456drfz.de
                [*]

                These authors contributed equally.

                Article
                NIHMS1987996
                10.1002/eji.202170126
                11115438
                34910301
                1f3858b1-c8fd-418f-9cf1-970d62e58e5d

                This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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