Methods in
Molecular Biology 2644
Oliver Friedrich
Daniel F. Gilbert Editors
Cell Viability
Assays
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, UK
For further volumes:
http://www.springer.com/series/7651
,For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series was
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biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by
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, Cell Viability Assays
Methods and Protocols
Second Edition
Edited by
Oliver Friedrich and Daniel F. Gilbert
Institute of Medical Biotechnology (MBT), Department of Chemical and Biological Engineering (CBI),
Friedrich-Alexander-University Erlangen-Nüremberg, Erlangen, Germany
,Editors
Oliver Friedrich Daniel F. Gilbert
Institute of Medical Biotechnology (MBT) Institute of Medical Biotechnology (MBT)
Department of Chemical and Biological Department of Chemical and Biological
Engineering (CBI) Engineering (CBI)
Friedrich-Alexander-University Friedrich-Alexander-University
Erlangen-Nüremberg Erlangen-Nüremberg
Erlangen, Germany Erlangen, Germany
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-0716-3051-8 ISBN 978-1-0716-3052-5 (eBook)
https://doi.org/10.1007/978-1-0716-3052-5
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part
of Springer Nature 2017, 2023
This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part
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The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.
,Obituary
I was deeply saddened to learn of the untimely and unexpected death of my co-editor and
long-time colleague, Dr Daniel Gilbert. He died at home in his sleep on the 25th of March,
2023, aged only 48 - much too young. I knew Daniel for 15 years. We were not only
colleagues, but also friends and shared a love of Australia, where we first met in 2008. Daniel
was a dedicated and passionate scientist as well as a smart inventor. He passed away during
the final stages of this book, his last scientific work and our last joint-project. Daniel will be
sorely missed, but his memory will live on through the pages of this book. Rest in peace,
Daniel.
Oliver Friedrich, on 27th March 2023
v
,Preface
Assessing cell viability as a measure for cell fitness under conditions of physiological and
pathophysiological stress as well as challenging conditions to cellular and tissue homeostasis
has become a universal bioanalytical task not only in life sciences and biomedical research but
more and more also in areas of biotechnology, environmental and clinical toxicology,
biomedical engineering, tissue engineering, and oncology, to name a few. As such, standar-
dized protocols and routines to assess various aspects of cellular homeostasis, viability, and
function are needed, their applicability toward specific tissues and generalization to other
cell entities and their limitations must be clarified, and potential combinations of different
readouts need to be discussed. Originally developed for classical in vitro 2D culture condi-
tions of cells interacting with stiff substrates as monolayers, recent developments in tissue
engineering or whole tissue applications in situ are calling out for reliable methods to be
applied to the next dimension for cells interacting with extracellular matrices in 3D. As such,
one of the major challenges in the coming years is to validate cell viability assays regarding
their upscaling from 2D to 3D and where limitations thereof have to be acknowledged.
Our first edition of Cell Viability Assays for the MiMB series in 2017, with its described
variety of assays, proved to be quite a success regarding its impact in the community. When
the publisher approached us to consider an updated second edition in 2021, the first edition
had already been accessed over 100,000 times.
For this updated edition, we therefore aimed to not only update and include new assays
but also to account for the ongoing 2D-3D development and planned for topics and assays
that targeted cell viability, mobility, and functionality of tissues and organs, natural or
bioartificial, in 3D. We approached renowned experts in the fields of 2D and 3D cell viability
assays and were able to secure 30 contributions spanning a wide range of viability and
functionality assays, from impedance spectroscopy to chemiluminescence, fluorescence, and
label-free optical detection methodologies, providing corresponding “Tips & Tricks”
regarding their applications and analyses strategies, a hallmark of the MiMB series. Apart
from “classical” cell viability aspects represented by “alive-dead” dichotomy, we also were
able to include several chapters that cover various aspects of cell function, e.g., electrical
excitability, contraction, and mobility, as well as cell adhesion or cell cycle analysis, among
others.
The first two-thirds of the chapters are grouped with a focus on 2D aspects of cell
viability assays while the remaining third is dedicated to the third dimension.
We thank all the authors who contributed to making this exciting new second edition of
the volume possible, and we are confident that it will provide a valuable resource to the
growing community of bioinspired life sciences, biomedical sciences, and biotechnology in
providing more standardized protocols to probe cells in various environments regarding
their “wellbeing.”
Institute of Medical Biotechnology (MBT), Department Oliver Friedrich
of Chemical and Biological Engineering (CBI) Daniel F. Gilbert
Friedrich-Alexander University
Erlangen-Nürnberg, Erlangen, Germany
vii
,Contents
Obituary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii
PART I SINGLE CELL ASSAYS IN 2D ENVIRONMENTS
1 Assaying Mitochondrial Respiration as an Indicator of Cellular Metabolism
and Fitness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Natalia Smolina, Aleksandr Khudiakov, and Anna Kostareva
2 The MTT Assay: A Method for Error Minimization and Interpretation
in Measuring Cytotoxicity and Estimating Cell Viability . . . . . . . . . . . . . . . . . . . . . 15
Mahshid Ghasemi, Sisi Liang, Quang Minh Luu,
and Ivan Kempson
3 Assaying Proliferation Characteristics of Cells Cultured Under Static
Versus Periodic Conditions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Daniel F. Gilbert, Oliver Friedrich, and Joachim Wiest
4 Network Reconstruction as a Novel High-Level Marker of Functional
Neuronal Viability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Jana Katharina Dahlmanns and Marc Dahlmanns
5 Assaying Mitochondrial Function by Multiparametric Flow Cytometry . . . . . . . . 65
Hannah C. Sheehan, Jonathan L. Tilly, and Dori C. Woods
6 High-Efficiency Single-Cell Electrical Impedance Spectroscopy . . . . . . . . . . . . . . . 81
Yongxiang Feng, Liang Huang, Peng Zhao, Fei Liang,
and Wenhui Wang
7 Cell Viability Multiplexing: Quantification of Cellular Viability by Barcode
Flow Cytometry and Computational Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Valentina Giudice, Victoria Fonseca, Carmine Selleri,
and Massimo Gadina
8 Average Rheological Quantities of Cells in Monolayers . . . . . . . . . . . . . . . . . . . . . . 123
Santanu Kumar Basu, Haider Dakhil, and Andreas Wierschem
9 Assaying Spontaneous Network Activity and Cellular Viability
Using Multi-Well Microelectrode Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Seline S. Choo, Jackson Y. Keever, Jasmine Brown,
Jenna D. Strickland, and Timothy J. Shafer
10 Quantitative Live-Cell Ca2+ Imaging During Isotropic Cell Stretch . . . . . . . . . . . 155
Ulrike Schöler, Anna-Lena Merten, Sebastian Schürmann,
and Oliver Friedrich
11 Assessment of Cell Viability in Electrically Excitable Muscle Cells
Through Intact Twitch Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Julian Bauer, Stewart I. Head, and Oliver Friedrich
12 Evaluating Cellular Viability by iTRAQ Proteomic Profiling . . . . . . . . . . . . . . . . . 193
Anne Poljak, Mark Raftery, and Patsie Polly
ix
,x Contents
13 Quantifying pH in Malaria Using pHluorin and Flow Cytometry . . . . . . . . . . . . . 211
Jeffrey Agyapong and Petra Rohrbach
14 Cell Membrane State, Permeability, and Elasticity Assessment for Single
Cells and Cell Ensembles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
Nicolas F€a rber, Simon V. Neidinger, and Christoph Westerhausen
15 Neutral Red Uptake Assay to Assess Cytotoxicity In Vitro . . . . . . . . . . . . . . . . . . . 237
Robim M. Rodrigues, Marth Stinckens, Gamze Ates,
and Tamara Vanhaecke
16 Digital Holographic Microscopy to Assess Cell Behavior. . . . . . . . . . . . . . . . . . . . . 247
Brad Bazow, Van K. Lam, Thuc Phan, Byung Min Chung,
George Nehmetallah, and Christopher B. Raub
17 Second Harmonic Generation Morphometry of Muscle Cytoarchitecture
in Living Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
Dominik Schneidereit, Stefanie Nübler, and Oliver Friedrich
18 Optimization of Cell Viability Assays for Drug Sensitivity Screens. . . . . . . . . . . . . 287
Peter Larsson and Toshima Z. Parris
19 Cellasys #8: A Microphysiometric Assay for Real-Time Cell Analysis
Within 24 Hours . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
Sebastian Eggert, Svitlana Stetsenko, and Joachim Wiest
20 Live Cell Adhesion, Migration, and Invasion Assays . . . . . . . . . . . . . . . . . . . . . . . . . 313
Jordi Pijuan, Anna Macià, and Anaı̈s Panosa
21 Cell Viability and Immunogenic Function of T Cells Loaded
with Nanoparticles for Spatial Guidance in Magnetic Fields . . . . . . . . . . . . . . . . . . 331
Felix Pfister, Christoph Alexiou, and Christina Janko
PART II SINGLE CELL ASSAYS IN 3D ENVIRONMENTS
22 Transwell In Vitro Cell Migration and Invasion Assays . . . . . . . . . . . . . . . . . . . . . . 349
Calvin R. Justus, Mona A. Marie, Edward J. Sanderlin,
and Li V. Yang
23 Calcium Imaging of Non-adherent Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361
Lydia J. Bye, Rocio K. Finol-Urdaneta, and David J. Adams
24 FUCCI Reporter Gene-Based Cell Cycle Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . 371
Lena Fischer and Ingo Thievessen
25 Cell Viability Assays for 3D Cellular Constructs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
Zachary Congress, Matthew Brovold, and Shay Soker
26 Toxicological Analysis by Assessment of Vascularization and Cell Viability
Using the Chicken’s Chorioallantoic Membrane (CAM Assay) . . . . . . . . . . . . . . . 403
Nadine Wiesmann, Jürgen Brieger, and Jonas Eckrich
27 Assessment of Tissue Viability by Functional Imaging
of Membrane Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423
Peter Kohl and Callum M. Zgierski-Johnston
, Contents xi
28 Multiplexed Viability Assays for High-Throughput Screening of Spheroids
of Multiple Sizes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435
Mohana Marimuthu and Thomas Gervais
29 Tracking Gut Motility in Organ and Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449
Peng Du, Vikram Joshi, and Arthur Beyder
30 Optimized Method of 3D Scaffold Seeding, Cell Cultivation,
and Monitoring Cell Status for Bone Tissue Engineering . . . . . . . . . . . . . . . . . . . . 467
Adrian Krolinski, Kai Sommer, Johanna Wiesner,
Oliver Friedrich, and Martin Vielreicher
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 481
