Cell Viability Assays: Methods and Protocols (Methods in Molecular Biology, 2644)

✍ Scribed by Oliver Friedrich (editor), Daniel F. Gilbert (editor)

• Publisher: Humana
• Year: 2023
• Tongue: English
• Leaves: 501
• Edition: 2nd ed. 2023
• Category: Library

✦ Synopsis

This updated edition explores assessing cell viability as a measure for cell fitness under conditions of physiological and patho-physiological stress as well as challenging conditions to cellular and tissue homeostasis, and accounts for the ongoing 2D-to-3D development with topics and assays that target cell viability, mobility, and functionality of tissues and organs, natural or bioartificial, in 3D. The book’s contents span a wide range of viability and functionality assays, from impedance spectroscopy to chemiluminescence, fluorescence and label-free optical detection methodologies. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. 
Authoritative and up-to-date, Cell Viability Assays: Methods and Protocols, Second Edition serves as a valuable resource to the growing community in bioinspired life sciences, biomedical sciences, and biotechnology by providing more standardized protocols to probe the “wellbeing” of cells in various environments.

✦ Table of Contents

Obituary
Preface
Contents
Contributors
Part I: Single Cell Assays in 2D Environments
Chapter 1: Assaying Mitochondrial Respiration as an Indicator of Cellular Metabolism and Fitness
Abbreviations
1 Introduction
2 Materials
2.1 Cell Culture Reagents and Consumables
2.2 XF24 Analyzer Assay Reagents and Consumables
2.3 Equipment Used
3 Methods
3.1 iPSC-CM Preparation for the Measurement of O2 Consumption Rates
3.2 C2C12 Cell Preparation for the Measurement of O2 Consumption Rates
3.3 Measurement of Cellular O2 Consumption Rates: Prior to the Day of Assay
3.4 Measurement of Cellular O2 Consumption Rates: Day of Assay
3.5 Data Analysis and Normalization
4 Notes
References
Chapter 2: The MTT Assay: A Method for Error Minimization and Interpretation in Measuring Cytotoxicity and Estimating Cell Via...
1 Introduction
2 Materials
2.1 General Equipment
2.2 Reagents
3 Methods
3.1 Making the Right Choice and Planning Ahead
3.2 Optimizing and Performing the Assay
3.3 Choosing the Optimum Assay Parameters, Data Analysis, and Interpretation
4 Notes
References
Chapter 3: Assaying Proliferation Characteristics of Cells Cultured Under Static Versus Periodic Conditions
1 Introduction
2 Materials
2.1 General Equipment
2.2 Biological Material
2.3 Solutions and Reagents
2.4 Software
2.5 Specific Hardware
3 Methods
3.1 Cell Culture
3.2 Cell Counting and Cell Chip Preparation
3.3 Long-Term Culture and Life-Cell Time-Lapse Fluorescence Imaging
4 Notes
References
Chapter 4: Network Reconstruction as a Novel High-Level Marker of Functional Neuronal Viability
1 Introduction
2 Materials
2.1 Preparation of Primary Cultures
2.1.1 Equipment
2.1.2 Solutions and Media
2.2 Fluorescence Live-Cell Imaging
2.3 Conversion of Raw Camera Images
2.4 Files for Network Reconstruction
3 Methods
3.1 Preparation of Primary Hippocampus Cultures
3.2 Fluorescence Live-Cell Imaging of Neuronal Network Activity
3.3 Extraction of Fluorescence Traces and Spike Estimation
3.4 Network Reconstruction
3.5 Network Topology Analysis and Network Activity Analysis
3.6 Statistical Comparisons Between Different Experimental Groups
4 Notes
References
Chapter 5: Assaying Mitochondrial Function by Multiparametric Flow Cytometry
1 Introduction
2 Materials
2.1 General Equipment
2.2 Reagents
2.3 Buffers
2.4 Sample Mitochondrial Stains and Control Reagents
2.5 Cell Culture Reagents (Cell Line-Specific)
2.6 Antibody Labeling Reagents
2.7 Reagents Used in Instrument Calibration
3 Methods
3.1 Mitochondrial Staining
3.2 Cell Lysis
3.3 Antibody Labeling for OMM (Optional)
3.4 Antibody Labeling for Fixed Mitochondria (Optional)
3.5 Mitochondrial Analysis by Flow Cytometry
3.6 Sorting of Mitochondria (Optional)
4 Notes
References
Chapter 6: High-Efficiency Single-Cell Electrical Impedance Spectroscopy
1 Introduction
2 Materials
2.1 Analyte Preparation
2.2 Device Fabrication
2.3 Impedance Measurement System
2.4 Software and Analysis
2.5 Clean Room Equipment
2.6 Experimental Equipment
3 Methods
3.1 Cell Preparation
3.2 Microfluidic Chip Design
3.3 Soft Lithography for Fabricating Microchannel
3.4 Lift-Off Procedure for Fabricating Sensing Electrodes
3.5 Microchip Bonding
3.6 Impedance Measurement System Configuration
3.7 Experimental Setup and Data Analysis
3.8 Measurement System Calibration and Verification
3.9 IFC Validation and Cell Classification
3.10 EIS Validation and Electrical Property Extraction
3.11 Complementarity of EIS and IFC
4 Notes
References
Chapter 7: Cell Viability Multiplexing: Quantification of Cellular Viability by Barcode Flow Cytometry and Computational Analy...
1 Introduction
2 Materials
2.1 FCB Buffers and Dyes
2.2 Samples
2.3 Instruments and Software
3 Methods
3.1 Sample Preparation
3.2 Viability Dye Staining
3.3 Sample Fixation and Permeabilization
3.4 Control Preparations
3.5 Nine-Sample Matrix Preparation (3 x 3)
3.6 Fluorescent Cell Barcoding
3.7 Acquisition and Compensation
3.8 Manual Gating Strategy
3.9 Computational Gating Strategy
3.10 Validation of FCB Data: Intra-Assay Variability
3.11 Validation of FCB Data: Inter-Assay Variability
3.12 Validation of FCB Data: Internal Bridge Control
4 Notes
References
Chapter 8: Average Rheological Quantities of Cells in Monolayers
1 Introduction
2 Materials
3 Methods
3.1 Cell Preparation
3.2 Cell Staining
3.3 Cell Monolayer Rheometry with a Modified Narrow-Gap Rheometer
3.4 Alignment of Upper and Lower Plates in Narrow-Gap Rheometer
3.5 Coating the Rheometer Plates
3.6 Cell Seeding on to the Lower Glass Plate of the Rheometer
3.7 Cell Coverage Detection
3.8 Measurement of Viscoelastic Properties of Cells
4 Notes
References
Chapter 9: Assaying Spontaneous Network Activity and Cellular Viability Using Multi-Well Microelectrode Arrays
1 Introduction
2 Materials
2.1 MEA Recording
2.2 Network Formation Assay and Acute Screening Assay
2.3 Viability: CellTiter-Blue
2.4 Viability: LDH Release (Acute)
2.5 Viability: Total Cellular LDH (Network Formation)
2.6 Viability: Impedance Measurement (See Note 3)
3 Methods
3.1 Coating MEA Plates with Poly-Ethyleneimine (PEI)
3.2 Culturing on MEA Plates
3.3 MEA Recording
3.4 Network Formation Assay
3.5 Acute Assay
3.6 Viability: CellTiter-Blue Assay
3.7 Viability: LDH Release Assay
3.8 Total Cellular LDH Assay
3.9 Viability: Electrical Impedance
3.10 Viability: Electrical Impedance (Establishing Media Blank Values)
4 Notes
References
Chapter 10: Quantitative Live-Cell Ca2+ Imaging During Isotropic Cell Stretch
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Cell Stretching Device
2.3 Buffers and Solutions
2.4 Laboratory Equipment
2.5 Fluorescence Microscope
2.6 Image Processing with Fiji
2.7 Data Processing Software
3 Methods
3.1 Preparation and Surface Modification of the PDMS Cell Stretching Chambers
3.2 BJ Cell Culturing
3.3 BJ Cell Seeding to the PDMS Chambers (One Day Before Cell Stretching)
3.4 Preparation of the Cal-520 Staining Solution and of the Test Chemicals
3.5 Functional Ca2+ Imaging During Global Stretch
3.6 Acquisition of the Images Required for Image Correction
3.7 Image Processing Using FIJI Software
3.8 Data Analysis
3.9 Accessing Cell Viability Based on the Response to Ionomycin
4 Notes
References
Chapter 11: Assessment of Cell Viability in Electrically Excitable Muscle Cells Through Intact Twitch Stimulation
1 Introduction
2 Materials
2.1 General Equipment
2.2 Reagents and Solutions
2.3 Muscle Cell Stimulation Pen (ClickTwitch)
2.4 Muscle Dissection
2.5 Enzymatic Single Muscle Cell Bioseparation
2.6 Cell Viability Assessment Through Electrical Stimulation
3 Methods
3.1 PDMS Coating of Petri Dishes
3.2 Muscle Cell Stimulation Pen (ClickTwitch) (Optional Step If No Commercial Pulse Stimulator Is to Be Used and DOY-Approach ...
3.3 Muscle Dissection
3.4 Enzymatic Single Muscle Fiber Dissociation
3.5 Cell Viability Assessment Through Electrical Stimulation
4 Notes
References
Chapter 12: Evaluating Cellular Viability by iTRAQ Proteomic Profiling
1 Introduction
2 Materials
2.1 Solutions and Reagents
2.2 Test Kits
2.3 General Equipment and Software
3 Methods
3.1 Example Protocol for Sample Extraction and Total Protein Assay (Day One)
3.2 iTRAQ 8-Plex Labeling Followed by Cation Exchange Cleanup of Sample (Day Two)
3.3 C18 Cleanup and Checking by MSMS to Ensure that the Labeling Has Worked
3.4 Tandem Mass Spectrometry of iTRAQ-Labeled Peptides Using 2D LCMSMS
3.5 Data Analysis: ProteinPilot
4 Notes
5 Conclusion
References
Chapter 13: Quantifying pH in Malaria Using pHluorin and Flow Cytometry
1 Introduction
2 Materials
2.1 Cell Cultures
2.2 Solutions and Reagents
2.3 General Equipment
2.4 Software
3 Methods
3.1 Parasite Culturing
3.2 Parasite Preparation
3.3 pH Calibration
3.4 pH Quantification
4 Notes
References
Chapter 14: Cell Membrane State, Permeability, and Elasticity Assessment for Single Cells and Cell Ensembles
1 Introduction
2 Materials
2.1 Solutions and Reagents
2.2 Cell Culture
2.3 Other Equipment
3 Methods
3.1 Laurdan Staining and Incubation
3.2 Permeability Assessment
3.3 Spectral Analysis of Laurdan with Two Filter Cubes and Permeability Measurement
3.4 Spectral Analysis of Laurdan with a Spectrometer
3.5 Acquisition of Elasticity Data
3.6 Microscopy Data Analysis
3.7 Spectrometer Data Analysis
3.8 Mechanical Data Analysis
4 Notes
References
Chapter 15: Neutral Red Uptake Assay to Assess Cytotoxicity In Vitro
1 Introduction
2 Materials
2.1 General Equipment
2.2 Reagents
3 Methods
3.1 HepG2 Cell Culture
3.2 Preparation of Test Chemicals
3.3 Chemical Exposure
3.4 Neutral Red Uptake Assay
3.5 Data Analysis
4 Notes
References
Chapter 16: Digital Holographic Microscopy to Assess Cell Behavior
1 Introduction
2 Materials
2.1 Imaging Systems
2.2 General Equipment
2.3 Solutions and Reagents
2.4 Biological Specimens
2.5 Software
3 Methods
3.1 Digital Holographic Microscopy of Live Cell Cultures
3.2 Phase Map Reconstruction
3.3 Phase Aberration Correction
3.4 Phase Unwrapping
3.5 Analysis of Cell Morphology and Motility
4 Notes
References
Chapter 17: Second Harmonic Generation Morphometry of Muscle Cytoarchitecture in Living Cells
1 Introduction
2 Materials
2.1 Optics Equipment
2.2 Software
2.3 General Laboratory Equipment
2.4 Solutions and Reagents
3 Methods
3.1 Coating Culture Dishes with Silicone Elastomer
3.2 Single Fiber Extraction
3.3 TDE Clearing
3.4 Aligning Excitation and Collection Objectives
3.5 Pre-Focusing a Sample Using Bright-Field Illumination
3.6 Placement of Filters and Dichroic Mirrors
3.7 Single Fiber 3D SHG Image Acquisition
3.8 Whole Muscle 3D SHG Image Acquisition
3.9 Exporting an Image Slice from a 3D Stack While Maintaining the Size Information Metadata
3.10 3D Reconstruction of an Image Stack
3.11 3D Reconstruction of a Mosaic of Image Stacks
3.12 Determining Mean Sarcomere Length in an Image
3.13 Determining the Major Orientation Axis in an Image
3.14 Determining Fiber CAS in an Image
3.15 Determining Fiber VD in an Image
4 Notes
References
Chapter 18: Optimization of Cell Viability Assays for Drug Sensitivity Screens
1 Introduction
2 Materials
2.1 Pharmaceutical Compounds
2.2 Cell Culture
2.3 Resazurin Reduction Cell Viability Assay
2.4 Data Analysis
3 Methods
3.1 Dilute Pharmaceutical Compounds
3.2 Cell Culture
3.3 Determine the Optimal Seeding Density
3.4 Estimate the Cell Doubling Time (Td)
3.5 Drug Sensitivity Screening
3.6 Determine Signal Dynamic Range Using Quality Control Metrics
3.7 Calculate Cell Viability (Microsoft Excel)
3.8 IC50 and GR50 Value Calculation (R/Bioconductor)
4 Notes
References
Chapter 19: Cellasys #8: A Microphysiometric Assay for Real-Time Cell Analysis Within 24 Hours
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment and Tools
2.3 Consumables
2.4 Reagent Setup
2.4.1 Reference Medium
2.4.2 Treatment Medium
3 Methods
3.1 Day Prior to Assay
3.1.1 Print and Fill Quality Control Document
3.1.2 Setup 6x IMOLA-IVD
3.1.3 Sterilize 6x IMOLA-IVD
3.1.4 Prepare Medium Flasks with Reference Medium
3.1.5 Prepare Medium Flasks with Treatment Medium
3.1.6 Setup 6x IMOLA-IVD with Medium
3.1.7 First Prefilling with Medium
3.1.8 Seed Cells onto BioChips
3.2 Assay Day
3.2.1 Second Prefilling with Medium (see Note 12)
3.2.2 Assemble BioChips with Fluidic Heads
3.2.3 Assemble BioChips into 6x IMOLA-IVD
3.2.4 Start Assay
3.3 Day After Assay
3.3.1 Stop the Experiment and Export the Data
3.3.2 Dismount and Clean
4 Notes
References
Chapter 20: Live Cell Adhesion, Migration, and Invasion Assays
1 Introduction
2 Materials
2.1 General Equipment and Consumables
2.2 Reagents and Solutions
3 Methods
3.1 Spreading Assay
3.2 Wound Healing Assay
3.3 Individual Cell Tracking Assay
3.4 Transwell Cell Migration/Invasion Assay
4 Notes
References
Chapter 21: Cell Viability and Immunogenic Function of T Cells Loaded with Nanoparticles for Spatial Guidance in Magnetic Fiel...
1 Introduction
2 Materials
2.1 General Equipment and Consumables
2.2 Antibodies
2.3 Buffers and Cell Culture Media
2.4 Staining Solutions
3 Methods
3.1 Isolation of CD3+ T Cells via IBA CD3 Fab-TACS Gravity Kit
3.2 Loading of T Cells with Magnetic Nanoparticles
3.3 Analysis of Cell Viability and Cell Cycle
3.4 Intracellular Cytokine Staining
3.5 Analysis of T Activation Markers and Cell Subsets
3.6 Analysis of Cell Proliferation
4 Notes
References
Part II: Single Cell Assays in 3D Environments
Chapter 22: Transwell In Vitro Cell Migration and Invasion Assays
1 Introduction
2 Materials
2.1 Cells
2.2 Media and Solutions
2.3 Transwell Inserts
2.4 Chemoattractant
2.5 Equipment
3 Methods
3.1 Cell Culture
3.2 Transwell Cell Migration Assay (see Fig. 1)
3.3 Transwell Cell Invasion Assay (see Fig. 2)
3.4 Colorimetric Staining for Migrated Adherent Cells: Crystal Violet
3.5 Fluorescent Staining for Migrated Adherent Cells-DAPI
3.6 Quantification of Migrated Adherent Cells
3.7 Quantification of Migrated Nonadherent Cells
4 Notes
References
Chapter 23: Calcium Imaging of Non-adherent Cells
1 Introduction
2 Materials
2.1 Materials
2.2 Reagent Stocks
2.3 Imaging Solutions
2.4 Cell Culture Medium
2.5 5% (w/v) Gelatin Solution
3 Methods
3.1 THP-1 Cell Culture
3.2 Loading Cells with Calcium Indicator
3.3 Cell Immobilization
3.4 Ca2+ Fluorescence Acquisition Experiments
4 Notes
References
Chapter 24: FUCCI Reporter Gene-Based Cell Cycle Analysis
1 Introduction
2 Materials
2.1 General Equipment
2.2 Cell Lines
2.3 Cell Culture Media
2.4 Solutions and Reagents
2.5 Lentiviral Plasmids
2.6 Analysis Software
3 Methods
3.1 Cell Culture, Harvesting, and Counting
3.2 Determination of Puromycin Concentration
3.3 Generation and Concentration of Lentivirus (Transfection of LentiX 293 T Cells)
3.4 Infection of Target Cells (Transduction of NIH/3T3 Cells)
3.5 Imaging
3.6 Image Evaluation
4 Notes
References
Chapter 25: Cell Viability Assays for 3D Cellular Constructs
1 Introduction
2 Materials
2.1 Solutions, Reagents, and Tissue Constructs
2.2 General Equipment
2.3 Software
2.4 CT3D Assay
2.5 Live/Dead Assay
2.6 AB Assay
3 Methods
3.1 CellTiter-Glo 3D (CT3D) Viability Assay
3.2 Live/Dead Assay
3.3 MTS Assay
3.4 AlamarBlue (AB) Assay
4 Notes
References
Chapter 26: Toxicological Analysis by Assessment of Vascularization and Cell Viability Using the Chicken´s Chorioallantoic Mem...
1 Introduction
2 Materials
2.1 General Equipment
2.2 Solutions and Reagents
2.3 Laboratory Equipment and Software
2.4 Cells and Biological Materials
2.5 Antibodies for Immunohistochemistry
3 Methods
3.1 Chicken Egg Development and Incubation
3.2 Tumor Cell Culture
3.3 Tumor Cell Inoculation onto the CAM
3.4 Administration of Therapeutic Agents to the Chicken Embryo
3.5 In Ovo Imaging with (Fluorescence) Microscopy
3.6 Ultrasonography in the CAM Assay
3.7 Preparation of Tumor, CAM, and Organs for Pathological Analysis
3.8 Preparation for Immunohistochemical Staining
3.9 Hematoxylin and Eosin (HE) Staining
3.10 Immunohistochemical Staining for Alpha-smooth Muscle Actin (a-SMA) and Cleaved Caspase-3
4 Notes
References
Chapter 27: Assessment of Tissue Viability by Functional Imaging of Membrane Potential
1 Introduction
2 Materials
2.1 Solutions and Reagents
2.2 Heart Extraction and Cannulation
2.3 Langendorff Perfusion
2.4 Optical Mapping
2.5 Data Analysis
3 Methods
3.1 Langendorff System Preparation (see Note 11)
3.2 Heart Extraction and Cannulation
3.3 Langendorff Perfusion and Optical Mapping
3.4 Data Analysis
4 Notes
References
Chapter 28: Multiplexed Viability Assays for High-Throughput Screening of Spheroids of Multiple Sizes
1 Introduction
2 Materials
2.1 Microchip
2.2 Cell Lines
2.3 Fluorescent Stains and Markers
2.4 Solutions and Reagents
2.5 General Equipment and Others
3 Methods
3.1 Development of PMMA Molds for Spheroid Chip
3.2 Fabrication of Spheroid Chip (See Note 4)
3.3 Fabrication of PMMA Holder for Spheroid Chip
3.4 Synthesis of Spheroids
3.5 Chemotherapeutic Drug Response Analysis
3.6 Spheroid Staining for Confocal Imaging
3.7 Spheroid Capture and Staining for FACS Analysis
4 Notes
References
Chapter 29: Tracking Gut Motility in Organ and Cultures
1 Introduction
2 Materials
2.1 General Material
2.2 Solutions and Reagents
2.3 Hardware and Software
2.4 Animal Models
3 Methods
3.1 Large Animal Surgery and Preparation
3.2 Small Animal Surgery and Organotypic Cultures
3.3 Video Acquisition
3.4 Image Processing
3.5 Contractility Frequency Measurements
4 Notes
References
Chapter 30: Optimized Method of 3D Scaffold Seeding, Cell Cultivation, and Monitoring Cell Status for Bone Tissue Engineering
1 Introduction
2 Materials
2.1 General Equipment
2.2 Solutions and Reagents
2.3 Labels
2.4 Software
2.5 Biological Material
2.6 Scaffolds
3 Methods
3.1 Pretreatment of Scaffolds
3.2 Cell Seeding and Cultivation
3.3 Cell Proliferation Assay
3.4 Alkaline Phosphatase Activity Assay
3.5 Cell Preparation for Microscopy
3.6 Multiphoton Microscopy
4 Notes
References
Index