Methods in molecular biology vol 1601 cell viability assays methods and protocols

Methods in Molecular Biology 1601 Daniel F Gilbert Oliver Friedrich Editors Cell Viability Assays 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 Cell Viability Assays Methods and Protocols Edited by Daniel F Gilbert Friedrich-Alexander University (FAU) Erlangen-Nürnberg, Institute of Medical Biotechnology, Erlangen, Germany Oliver Friedrich Friedrich-Alexander University (FAU) Erlangen-Nürnberg, Institute of Medical Biotechnology, Erlangen, Germany Editors Daniel F Gilbert Friedrich-Alexander University (FAU)   Erlangen-Nürnberg Institute of Medical Biotechnology Erlangen, Germany Oliver Friedrich Friedrich-Alexander University (FAU)   Erlangen-Nürnberg Institute of Medical Biotechnology Erlangen, Germany ISSN 1064-3745     ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-6959-3    ISBN 978-1-4939-6960-9 (eBook) DOI 10.1007/978-1-4939-6960-9 Library of Congress Control Number: 2017936200 © 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 In vitro assessment of cellular viability has become a generic approach in addressing a vast range of biological questions in many areas of biomedical research The spectrum of available cell viability indicators assessing individual physiological, structural, or functional parameters is large and is continuously increasing with the availability and optimization of new or existing technologies Depending on the number and diversity of employed fitness indicators, a cell viability assay can generate fitness phenotypes of varying complexity: when a single indicator is used, the information provided on the cellular condition is very limited, potentially resulting in poor dataset concordance, whereas when various indicators are employed, e.g., in a multiplexing approach, combining different methods in one experiment, cellular fitness is reflected more comprehensively, allowing for decreased interassay variability and increased reproducibility of experimental results While cell-based viability screening is typically carried out using simple and single indicator-based approaches, a paradigm shift toward more advanced methods generating complex cell fitness phenotype readouts is currently taking over as indicated by an increasing availability of protocols describing multiparameter assaying techniques This book is intended to provide an overview and to discuss the strengths and pitfalls of commonly used cell fitness indicators We aim to give an in-depth view of protocols that are used in the classical cell-based viability screening approach and to provide experimental methods for advanced cell viability assaying strategies, including evaluation of e.g cellular transporter activity, intracellular calcium signaling, electrical network activity, synaptic vesicle recycling or ligand-gated ion channel function In this volume, we cover biochemical, fluorescence and luminescence-based strategies as well as computational and label-free methodologies for assaying cellular viability by means of e.g viscoelastic properties, impedance and multiphoton microscopy The biological samples used in the described approaches cover a broad range of specimen including conventional culture models, stem and primary cells as well as parasites These chapters address an interdisciplinary audience, including graduate students, postdoctoral fellows, and scientists in all areas of biomedical research As the concept of this series is meant to shed light into the sometimes tiny “tips and tricks” that decide over the success or flaw of biological experiments, we hope that the chapters will provide useful hints to the community Erlangen, Germany  Daniel F. Gilbert Oliver Friedrich v Contents Preface v Contributors ix   Basic Colorimetric Proliferation Assays: MTT, WST, and Resazurin Konstantin Präbst, Hannes Engelhardt, Stefan Ringgeler, and Holger Hübner   Assaying Cellular Viability Using the Neutral Red Uptake Assay Gamze Ates, Tamara Vanhaecke, Vera Rogiers, and Robim M Rodrigues   Assessment of Cell Viability with Single-, Dual-, and Multi-­Staining Methods Using Image Cytometry Leo Li-Ying Chan, Kelsey J McCulley, and Sarah L Kessel   High-Throughput Spheroid Screens Using Volume, Resazurin Reduction, and Acid Phosphatase Activity Delyan P Ivanov, Anna M Grabowska, and Martin C Garnett   A Protocol for In Vitro High-Throughput Chemical Susceptibility Screening in Differentiating NT2 Stem Cells Ann-Katrin Menzner and Daniel F Gilbert   Ferroptosis and Cell Death Analysis by Flow Cytometry Daishi Chen, Ilker Y Eyupoglu, and Nicolai Savaskan   Assaying Mitochondrial Respiration as an Indicator of Cellular Metabolism and Fitness Natalia Smolina, Joseph Bruton, Anna Kostareva, and Thomas Sejersen   An ATP-Based Luciferase Viability Assay for Animal African Trypanosomes Using a 96-Well Plate Keisuke Suganuma, Nthatisi Innocentia Molefe, and Noboru Inoue  9 SYBR® Green I-Based Fluorescence Assay to Assess Cell Viability of Malaria Parasites for Routine Use in Compound Screening Maria Leidenberger, Cornelia Voigtländer, Nina Simon, and Barbara Kappes 10 Screening Applications to Test Cellular Fitness in Transwell® Models After Nanoparticle Treatment Bastian Christ, Christina Fey, Alevtina Cubukova, Heike Walles, Sofia Dembski, and Marco Metzger 11 Assays for Analyzing the Role of Transport Proteins in the Uptake and the Vectorial Transport of Substances Affecting Cell Viability Emir Taghikhani, Martin F Fromm, and Jörg König 12 Metabolite Profiling of Mammalian Cell Culture Processes to Evaluate Cellular Viability Isobelle M Evie, Alan J Dickson, and Mark Elvin vii 19 27 43 61 71 79 89 97 111 123 137 viii Contents 13 Assaying Spontaneous Network Activity and Cellular Viability Using Multi-well Microelectrode Arrays Jasmine P Brown, Brittany S Lynch, Itaevia M Curry-Chisolm, Timothy J Shafer, and Jenna D Strickland 14 Quantitative Ratiometric Ca2+ Imaging to Assess Cell Viability Oliver Friedrich and Stewart I Head 15 Functional Viability: Measurement of Synaptic Vesicle Pool Sizes Jana K Wrosch and Teja W Groemer 16 Phenotyping Cellular Viability by Functional Analysis of Ion Channels: GlyR-Targeted Screening in NT2-N Cells Katharina Kuenzel, Sepideh Abolpour Mofrad, and Daniel F Gilbert 17 Systematic Cell-Based Phenotyping of Missense Alleles Aenne S Thormählen and Heiko Runz 18 Second Harmonic Generation Microscopy of Muscle Cell Morphology and Dynamics Andreas Buttgereit 19 Assessment of Population and ECM Production Using Multiphoton Microscopy as an Indicator of Cell Viability Martin Vielreicher and Oliver Friedrich 20 Average Rheological Quantities of Cells in Monolayers Haider Dakhil and Andreas Wierschem 21 Measurement of Cellular Behavior by Electrochemical Impedance Sensing Simin Öz, Achim Breiling, and Christian Maercker 22 Nano-QSAR Model for Predicting Cell Viability of Human Embryonic Kidney Cells Serena Manganelli and Emilio Benfenati 153 171 195 205 215 229 243 257 267 275 Index 291 Contributors Sepideh Abolpour Mofrad  •  Institute of Medical Biotechnology, Friedrich-Alexander-­ Universität Erlangen-Nürnberg, Erlangen, Germany Gamze Ates  •  Department of In Vitro Toxicology and Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Brussels, Belgium Emilio Benfenati  •  Department of Environmental Health Sciences, Laboratory of Environmental Chemistry and Toxicology, IRCCS—Istituto di Ricerche Farmacologiche “Mario Negri”, Milan, Italy Achim Breiling  •  DKFZ ZMBH Alliance, Division of Epigenetics, German Cancer Research Center, Heidelberg, Germany Jasmine P. Brown  •  Integrated Systems Toxicology Division, NHEERL, US EPA, NC, USA Joseph Bruton  •  Karolinska Institutet, Stockholm, Sweden Andreas Buttgereit  •  Institute of Medical Biotechnology, Friedrich-Alexander-­Universität Erlangen-Nürnberg, Erlangen, Germany Leo Li-Ying Chan  •  Department of Technology R&D, Nexcelom Bioscience LLC, Lawrence, MA, USA Daishi Chen  •  Translational Cell Biology and Neurooncology Laboratory of the Universitätsklinikum Erlangen (UKER), Friedrich-Alexander University of Erlangen– Nürnberg (FAU), and Department of Neurosurgery of the Universitätsklinikum Erlangen, Universitätsklinikum Erlangen (UKER), Friedrich-Alexander University of Erlangen – Nürnberg (FAU), Erlangen, Germany Bastian Christ  •  Translational Center Würzburg “Regenerative Therapies for Oncology and Musculosceletal Diseases”, Branch of Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, Würzburg, Germany Alevtina Cubukova  •  Translational Center Würzburg “Regenerative Therapies for Oncology and Musculosceletal Diseases”, Branch of Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, Würzburg, Germany Itaevia M. Curry-Chisolm  •  Integrated Systems Toxicology Division, NHEERL, US EPA, NC, USA Haider Dakhil  •  Institute of Fluid Mechanics, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany; Faculty of Engineering, University of Kufa, Najaf, Iraq Sofia Dembski  •  Chair Tissue Engineering and Regenerative Medicine, University Hospital Würzburg, Würzburg, Germany; Fraunhofer Institute for Silicate Research ISC, Würzburg, Germany Alan J. Dickson  •  Faculty of Life Sciences, The University of Manchester, Manchester, UK Mark Elvin  •  Faculty of Life Sciences, The University of Manchester, Manchester, UK Hannes Engelhardt  •  Institute of Bioprocess Engineering, Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany Isobelle M. Evie  •  Faculty of Life Sciences, The University of Manchester, Manchester, UK ix x Contributors Ilker Y. Eyupoglu  •  Translational Cell Biology and Neurooncology Laboratory of the Universitätsklinikum Erlangen (UKER), Friedrich-Alexander University of Erlangen– Nürnberg (FAU), and Department of Neurosurgery of the Universitätsklinikum Erlangen, Universitätsklinikum Erlangen (UKER), Friedrich-Alexander University of Erlangen – Nürnberg (FAU), Erlangen, Germany Christina Fey  •  Chair Tissue Engineering and Regenerative Medicine, University Hospital Würzburg, Würzburg, Germany Oliver Friedrich  •  Friedrich-Alexander University (FAU) Erlangen-Nürnberg, Institute of Medical Biotechnology, Erlangen, Germany Martin F. Fromm  •  Department of Clinical Pharmacology and Clinical Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, Friedrich-­Alexander-­Universität Erlangen-Nürnberg, Erlangen, Germany Martin C. Garnett  •  School of Pharmacy, University of Nottingham, Nottingham, UK Daniel F. Gilbert  •  Friedrich-Alexander University (FAU) Erlangen-Nürnberg, Institute of Medical Biotechnology, Erlangen, Germany Anna M. Grabowska  •  Cancer Biology, Division of Cancer and Stem Cells, School of Medicine, Queen’s Medical Centre, University of Nottingham, Nottingham, UK Teja W. Groemer  •  Department of Psychiatry and Psychotherapy, Friedrich-Alexander University of Erlangen-Nuremberg, Erlangen, Germany Stewart I. Head  •  School of Medical Sciences (SOMS), University of New South Wales (UNSW), Sydney, NSW, Australia Holger Hübner  •  Institute of Bioprocess Engineering, Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany Noboru Inoue  •  National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido, Japan Delyan P. Ivanov  •  Cancer Biology, Division of Cancer and Stem Cells, School of Medicine, Queen’s Medical Centre, University of Nottingham, Nottingham, UK Barbara Kappes  •  Institute of Medical Biotechnology, University of Erlangen-Nürnberg, Erlangen, Germany Sarah L. Kessel  •  Department of Technology R&D, Nexcelom Bioscience LLC, Lawrence, MA, USA Jưrg Kưnig  •  Department of Clinical Pharmacology and Clinical Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, Friedrich-Alexander-­ Universität Erlangen-Nürnberg, Erlangen, Germany Anna Kostareva  •  ITMO University, Saint Petersburg, Russia Katharina Kuenzel  •  Institute of Medical Biotechnology, Friedrich-Alexander-­Universität Erlangen-Nürnberg, Erlangen, Germany Maria Leidenberger  •  Institute of Medical Biotechnology, University of Erlangen-­ Nürnberg, Erlangen, Germany Brittany S. Lynch  •  Integrated Systems Toxicology Division, NHEERL, US EPA, NC, USA Christian Maercker  •  Esslingen University of Applied Sciences, Esslingen am Neckar, Germany; German Cancer Research Center (DKFZ), Genomics and Proteomics Core Facilities, Heidelberg, Germany Serena Manganelli  •  Department of Environmental Health Sciences, Laboratory of Environmental Chemistry and Toxicology, IRCCS—Istituto di Ricerche Farmacologiche “Mario Negri”, Milan, Italy Contributors xi Kelsey J. McCulley  •  Department of Technology R&D, Nexcelom Bioscience LLC, Lawrence, MA, USA Ann-Katrin Menzner  •  Department of Internal Medicine 5, University Medical Center Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Marco Metzger  •  Translational Center Würzburg “Regenerative Therapies for Oncology and Musculosceletal Diseases”, Branch of Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, Würzburg, Germany; Chair Tissue Engineering and Regenerative Medicine, University Hospital Würzburg, Würzburg, Germany Nthatisi Innocentia Molefe  •  National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido, Japan Simin Ưz  •  German Cancer Research Center (DKFZ), Epigenomics and Cancer Risk Factors, Heidelberg, Germany Konstantin Präbst  •  Institute of Bioprocess Engineering, Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany Stefan Ringgeler  •  Institute of Bioprocess Engineering, Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany Robim M. Rodrigues  •  Department of In Vitro Toxicology and Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Brussels, Belgium Vera Rogiers  •  Department of In Vitro Toxicology and Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Brussels, Belgium Heiko Runz  •  Institute of Human Genetics, University of Heidelberg, Heidelberg, Germany; Molecular Medicine Partnership Unit (MMPU), University of Heidelberg/ EMBL, Heidelberg, Germany; Department of Genetics and Pharmacogenomics, Merck Research Laboratories, Boston, MA, USA Nicolai Savaskan  •  Translational Cell Biology and Neurooncology Laboratory of the Universitätsklinikum Erlangen (UKER), Friedrich-Alexander University of Erlangen– Nürnberg (FAU), and Department of Neurosurgery of the Universitätsklinikum Erlangen, Universitätsklinikum Erlangen (UKER), Friedrich-Alexander University of Erlangen – Nürnberg (FAU), Erlangen, Germany; BiMECON Ent., www.savaskan.net, Berlin, Germany Thomas Sejersen  •  Karolinska Institutet, Stockholm, Sweden Timothy J. Shafer  •  Integrated Systems Toxicology Division, NHEERL, US EPA, NC, USA Nina Simon  •  Institute of Medical Biotechnology, University of Erlangen-Nürnberg, Erlangen, Germany Natalia Smolina  •  Karolinska Institutet, Stockholm, Sweden; Federal Almazov North-West Medical Research Centre, Russia Jenna D. Strickland  •  Axion Biosystems, Atlanta, GA, USA; Department of Pharmacology and Toxicology, Michigan State University, MI, USA Keisuke Suganuma  •  National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido, Japan Emir Taghikhani  •  Department of Clinical Pharmacology and Clinical Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, Friedrich-­ Alexander-­Universität Erlangen-Nürnberg, Erlangen, Germany Aenne S. Thormählen  •  Institute of Human Genetics, University of Heidelberg, Heidelberg, Germany; Molecular Medicine Partnership Unit (MMPU), University of Heidelberg/EMBL, Heidelberg, Germany Nano-QSAR for Cell Viability Predictions 285 where yobs and ypred are the observed and predictive values while yobs and y pred are the mean values of the observed and predictive values respectively; Q å( y =1å( y obs - y pred ) obs - y) (4) where y means average activity value of the training set, whereas yobs and ypred represent observed and LOO-predicted activity values of the training set å( y ) - ycalc ) - yobs calc F= p å( y obs n - p -1 (5) where n is the number of compounds and p is the number of predictor variables s= PRESS n (6) where PRESS represents the predicted residual sum of squares and n is number of compounds PRESS is given by the expression: PRESS = å ( yobs - y pred ) (7) According to the criteria indicated by Golbraikh and Tropsha [35] revised by Tropsha [36], a model has high predictive power if the following conditions are fulfilled: Q2 > 0.5 R2 > 0.6 (R2 −  R02 )/R2