Mechanobiology: Methods and Protocols

Preface
Contents
Contributors
Part I: Quantifying the Mechanical Properties of Cells and Adhesion Proteins
	Chapter 1: Measuring Cell Mechanical Properties Using Microindentation
		1 Introduction
		2 Materials
			2.1 Consumable
			2.2 Microscope
			2.3 Micromanipulation-Related Equipment
			2.4 Water Reservoir
			2.5 Micropipette Manufacturing
		3 Methods
			3.1 Fabrication of Cell-Holding Micropipettes
			3.2 Fabrication of Flexible Micropipettes and Microindenters
			3.3 Calibrate a Reference Microindenter Using a Commercial Probe
			3.4 Calibration of the Bending Stiffness
			3.5 Automated Detection of the Force and Indentation, and Feedback Loop
			3.6 Microindentation of Nonadherent Cells
			3.7 Microindentation of Adherent Cells
			3.8 Microindentation Using an Activating Microbead
			3.9 End of Experiments
			3.10 Data and Statistical Analysis
		4 Notes
		References
	Chapter 2: In Situ Measurements of Cell Mechanical Properties Using Force Spectroscopy
		1 Introduction
		2 Materials
		3 Methods
			3.1 Cell Culture Preparation
			3.2 Probe Calibration
			3.3 Optical Probe Alignment
			3.4 Topographical Probe Alignment
			3.5 Nanoindentation Measurements
				3.5.1 Experimental Parameters Optimization
				3.5.2 Acquisition of Force Spectra
				3.5.3 Handling Tilted Baseline
			3.6 Force-Spectra-Based Imaging
				3.6.1 Mechanical Mapping of  Cell
				3.6.2 High-Resolution Nanoindentation Imaging of Specific Features
			3.7 Duration of Experimental Sessions
			3.8 Data Analysis
				3.8.1 Curve Smoothing
				3.8.2 Baseline Detection by Force-Curve Tail Analysis
				3.8.3 Baseline Detection by Force Distribution Analysis
				3.8.4 Contact Point Alignment by Point of First Repulsive Force
				3.8.5 Contact Point Alignment by Smoothed Slope Method
				3.8.6 Calculate Indentation Depth
				3.8.7 Fitting Contact Model
		4 Notes
		References
	Chapter 3: Quantification of Apparent Membrane Tension and Membrane-to-Cortex Attachment in Animal Cells Using Atomic Force Mi...
		1 Introduction
			1.1 Rationale of Static Tether Pulling
			1.2 Rationale of Dynamic Tether Pulling
		2 Materials
		3 Methods
			3.1 Sample Preparation: Animal Cells
			3.2 AFM Preparation: Calibration and Functionalization
			3.3 Measurements
				3.3.1 Static Tether Pulling
				3.3.2 Dynamic Tether Pulling
			3.4 Data Analysis
				3.4.1 Processing
				3.4.2 Plotting/Fitting
		4 Notes
		References
	Chapter 4: Characterizing the Biophysical Properties of Adhesive Proteins in Live Cells Using Single-Molecule Atomic Force Mic...
		1 Introduction
		2 Materials
			2.1 AFM Equipment
			2.2 Cell Culture
			2.3 AFM Sample Preparation
		3 Methods
			3.1 Tip Blunting and Spring Constant Calculation
			3.2 Cantilever Functionalization
			3.3 Immobilization of Biotinylated Ecad on AFM Cantilevers
			3.4 Cell Culture
			3.5 Cell Culture on Glass Coverslips
			3.6 AFM Experiment
			3.7 Data Analysis
		4 Notes
		References
Part II: Exposing Cells to External Mechanical Forces
	Chapter 5: Application of Shear Stress to Endothelial Cells Using a Parallel Plate Flow Chamber
		1 Introduction
		2 Materials
			2.1 Collagen Coating and Cell Seeding
			2.2 Parallel Plate Flow Chamber Set  Up
			2.3 Endothelial Cell Flow Adaptation Analysis
		3 Methods
			3.1 Collagen-Coated Microscope Slide Preparation
			3.2 Endothelial Cell Seeding
			3.3 Parallel Plate Flow Chamber Setup (Fig. 1)
			3.4 Endothelial Cell Adaptation Analysis via Immunofluorescence (Fig. 3)
			3.5 Cleaning and Sterilizing the Flow Chamber
		4 Notes
		References
	Chapter 6: Cell Stretcher Assay to Analyze Mechanoresponses to Cyclic Stretching
		1 Introduction
		2 Materials
			2.1 A7r5 Smooth Muscle Cell Culture
			2.2 Elastomer Substrates
			2.3 Preparation of Elastomer Chambers
			2.4 Cell Stretching and Analysis of Cytoskeletal Mechanoresponse
		3 Methods
			3.1 Cell Culture and Passaging
			3.2 Fabrication of Elastomer Chambers
			3.3 Mounting of Elastomer Substrates and Surface Coating
			3.4 Cell Seeding and Pre-incubation
			3.5 Cell Stretcher Assay
			3.6 Fixation and Staining of the Actin Cytoskeleton
		4 Notes
		References
	Chapter 7: Two-Point Optical Manipulation of Cell Junctions in the Early Epithelium of the Drosophila Embryo
		1 Introduction
		2 Materials
			2.1 Safety Measures
			2.2 Lasers and Calibration
			2.3 Optical  Path
			2.4 Drosophila Embryo Sample
		3 Methods
			3.1 Calibration of the Optical Trap Position
			3.2 Recording of a Calibration Movie
			3.3 Position Interpolation with Image Analysis
			3.4 Two-Point Optical Manipulation
			3.5 Selecting the Optical Trap Position on the Screen
			3.6 Sample Preparation
			3.7 Optical Manipulation of Junctions in the Epithelium of the Drosophila Embryo
			3.8 Extending/Shrinking the Junction
			3.9 Feedback Control for Motorized Stage
		4 Notes
		References
Part III: Quantitative Characterization of Cell Mechanosensing
	Chapter 8: A Microfluidic-Like System (MLS) to Grow, Image, and Quantitatively Characterize Rigidity Sensing by Plant´s Roots ...
		1 Introduction
		2 Materials
			2.1 Culture Media
			2.2 Microfluidic Chips
			2.3 Chip Bonding
			2.4 Seedling Preparation
			2.5 Manipulation
		3 Methods
			3.1 Seed Sterilization
			3.2 Seed Stratification
			3.3 Chip Preparation
			3.4 Chip Bonding and Solid Media Filling
			3.5 Plant Seeding
			3.6 Plant Seeding with the Tip Technique
			3.7 Plantlet Culture
			3.8 Video Microscopy
		4 Notes
		References
	Chapter 9: Photoresponsive Hydrogels for Studying Mechanotransduction of Cells
		1 Introduction
		2 Materials
			2.1 o-Nitrobenzyl Bis-acrylate (o-NBbA) Photocleavable Cross-Linker
			2.2 1D-Fibronectin-Coated Photoresponsive Gel: Preparation
			2.3 1D-Fibronectin-Coated Photoresponsive Gel: Characterization
			2.4 Instruments
		3 Methods
			3.1 Abbreviations
			3.2 Synthesis of o-NBbA
				3.2.1 2-Nitro-4-ethyl Aniline (S2)
				3.2.2 4-Ethyl-3-nitrophenol (S3)
				3.2.3 Tert-butyl 2-(4-ethyl-3-nitrophenoxy)acetate (S4)
				3.2.4 Tert-butyl 2-(4-(1-bromoethyl)-3-nitrophenoxy)acetate (S5)
				3.2.5 2-(4-(1-bromoethyl)-3-nitrophenoxy)ethan-1-ol (S6)
				3.2.6 1-(4-(2-hydroxyethoxy)-2-nitrophenyl)ethan-1-ol (S7)
				3.2.7 1-(4-(2-(acryloyloxy)ethoxy)-2-nitrophenyl)ethyl Acrylate (o-NBMA)
			3.3 Silane-Glutaraldehyde Activation of Glass Bottom Culture Dishes
			3.4 Preparation of Photoresponsive Polyacrylamide  Gels
			3.5 Preparation of 1D-Fibronectin Micropatterns on the Gel Surface
			3.6 Fabrication of Stepwise Stiffness Gradients by Controlled UV Exposure
			3.7 Stiffness Characterization by Bead Indentation (Calibration of Photoirradiation time vs. photocleavage)
			3.8 Characterization of Fibronectin Density by Immunofluorescence Detection
		4 Notes
		References
	Chapter 10: Analysing Mechanically Evoked Currents at Cell-Substrate Junctions
		1 Introduction
		2 Materials
			2.1 Fabricating Negative Masters to Cast Arrays
			2.2 Casting Pillar Substrates
			2.3 Sample Preparation
			2.4 Whole-Cell Patch-Clamp Electrophysiology
		3 Methods
			3.1 Design and Obtain Positive Masters
			3.2 Preparing Negative Masters
			3.3 Casting Pillar Arrays
			3.4 Preparing Cell Samples on Pillar Arrays
			3.5 Measuring MA Currents
			3.6 Analysis
				3.6.1 Characterising Currents
				3.6.2 Calculating Pillar Deflection
		4 Notes
		References
	Chapter 11: Quantifying Strain-Sensing Protein Recruitment During Stress Fiber Repair
		1 Introduction
		2 Materials
			2.1 Cells and Culture Media
			2.2 Constructs
			2.3 Transfection Equipment
			2.4 Microscope
		3 Methods
			3.1 Transfections (see Note 6)
			3.2 Image Acquisition
			3.3 Image Analysis
		4 Notes
		References
Part IV: Measuring Forces Applied by Cells on the Extracellular Matrix
	Chapter 12: Quantification of Invadopodia Formation and Matrix Degradation Activity
		1 Introduction
		2 Materials
		3 Methods
			3.1 Preparation of Alexa Fluor 488-Gelatin Coated Plates
			3.2 Plating Cancer Cells on the Labeled Gelatin Matrix
				3.2.1 Experimental Approach for Screening Different Cell Lines for Invadopodia Formation Using the Gelatin Degradation Assay
				3.2.2 Experimental Approach for Testing the Effect of Specific Gene Knockdown on Invadopodia Formation Using Gelatin Degradati...
				3.2.3 Experimental Approach for Screening Inhibitors of Invadopodia Formation Using Gelatin Degradation Assay
				3.2.4 Application of Microscopy-Based Image Processing for Quantification of Invadopodia Formation and Gelatin Degradation (in...
		4 Notes
		References
	Chapter 13: Measuring Cellular Traction Forces with Micropillar Arrays
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Solutions and Media
			2.3 PDMS Mixture
		3 Methods
			3.1 Pillar Fabrication
			3.2 Experiment Setup
				3.2.1 ECM Coating by Immersion
				3.2.2 ECM Coating by Stamping
			3.3 Visualizing the Cells
			3.4 Pillar Displacement Analysis Using ImageJ/FIJI (See Note 3)
				3.4.1 Prepare the File for Analysis Using ImageJ
				3.4.2 Define the ``Noise´´ and ``Reference´´ Pillars and Record their Positions
				3.4.3 Analyze the Noise and Reference Pillar Displacements Using MATLAB
				3.4.4 Analyze the Pillars That Were Displaced by Cells
		4 Notes
		References
	Chapter 14: Imaging Cell Adhesive Force at the Single Molecule Level
		1 Introduction
		2 Materials
			2.1 DNA Strands and an Optional PNA Strand
			2.2 Chemicals and Materials
				2.2.1 Chemicals Required for ITS Synthesis
				2.2.2 Consumables Required for ITS Surface-Immobilization and Cell Culture
				2.2.3 Equipment and Devices
		3 Methods
			3.1 Conjugation of RGD Peptide Ligand to the Upper DNA Strand (See Note 1)
			3.2 ITS Assembly
			3.3 ITS Immobilization on Glass Surfaces
			3.4 Cell Plating on the ITS Surface
				3.4.1 Cell Culture
				3.4.2 Cell Plating
			3.5 Imaging Cell Adhesive Force in a Cumulative  Mode
			3.6 Imaging Cell Adhesive Force in a Real-Time and Single Molecule  Mode
		4 Notes
		References
Part V: Force Measurement Inside Cells
	Chapter 15: Multiplexed Molecular Tension Sensor Measurements Using PIE-FLIM
		1 Introduction
			1.1 Tension Sensor Multiplexing
			1.2 Pulsed Interleaved Excitation (PIE)
		2 Materials
			2.1 Transient Expression of Tension Sensor Constructs
			2.2 Stable Expression of Tension Sensor Constructs
			2.3 Live Cell Microscopy and Image Acquisition with PIE-FLIM
			2.4 Data Analysis and Evaluation
		3 Methods
			3.1 Transient Transfection of Tension Sensor Constructs
			3.2 Transduction of Tension Sensor Constructs
				3.2.1 Virus Production
				3.2.2 Transduction
			3.3 Live Cell Microscopy and Image Acquisition with PIE-FLIM
				3.3.1 Microscope and Software Setup
				3.3.2 FLIM Measurement Routine
			3.4 Data Analysis and Evaluation
				3.4.1 Fitting Fluorescence Lifetimes Using the SymPhoTime Software
				3.4.2 Calculation of FRET Efficiencies
				3.4.3 Data Interpretation
		4 Notes
		References
	Chapter 16: Visualizing Neurons Under Tension In Vivo with Optogenetic Molecular Force Sensors
		1 Introduction
			1.1 FRET-Force Measurements
			1.2 Organization of the Protocol
		2 Materials
			2.1 Reagents and Consumables
			2.2 Equipment
			2.3 Software
			2.4 Animal Strains
			2.5 Equipment Setup
				2.5.1 Confocal Laser Scanning Microscope
				2.5.2 Standard Setup
		3 Methods
			3.1 Calibrating the Collection Efficiency of the Photodetector
				3.1.1 Determination of Collection Efficiency
				3.1.2 Analysis
				3.1.3 Optional: Determination of Pixel Noise for Each Photodetector
				3.1.4 Analysis
			3.2 Age Synchronizing Animals Before Imaging
			3.3 Mounting Animals for Imaging
			3.4 Imaging
				3.4.1 Quantifying Spectral Contaminants (Bleedthrough, Crosstalk)
			3.5 Analysis
		4 Notes
		References
	Chapter 17: Single-Cell Quantification of the Mechanical Stability of Cell-Cell Adherens Junction Using Glass Micropipettes
		1 Introduction
		2 Materials
		3 Methods
			3.1 Glass Micropipette Preparation
				3.1.1 Micropipette Pulling
				3.1.2 Micropipette Tip Cutting
				3.1.3 Tip Filling and Blocking
			3.2 Force Calibration
				3.2.1 Force Calibration of the Transverse Magnetic Tweezers
				3.2.2 Calibrate a Standard Glass Micropipette
				3.2.3 Calibrate the Working Glass Micropipette
			3.3 Prepare Probing Microbeads
			3.4 Chamber Preparation
				3.4.1 Chamber Assembly
				3.4.2 Cell Planting
			3.5 Single-Cell Adhesion Force Measurement
				3.5.1 System Setup
				3.5.2 Adhesion Force Measurement
		4 Notes
		References
Part VI: Probing Cell Signaling and Gene Expression in Response to Force
	Chapter 18: Using Micropatterned Supported Lipid Bilayers to Probe the Mechanosensitivity of Signaling Receptors
		1 Introduction
		2 Materials
			2.1 SUV Preparation
			2.2 Substrate Micropatterning
			2.3 Live-Cell Imaging
		3 Methods
			3.1 Substrate Micropatterning
			3.2 Prepare Micropatterned Substrate of Polymers and   SLBs
			3.3 Protein Functionalization
			3.4 Live-Cell Imaging
		4 Notes
		References
	Chapter 19: Monitoring Mechano-Regulation of Gene Expression by RNA Sequencing
		1 Introduction
			1.1 Method Overview
		2 Materials
		3 Methods
		4 Notes
		References
	Chapter 20: Testing the Role of Focal Adhesion Kinase (FAK) in Topography-Mediated Stem Cell Differentiation by Inhibiting FAK...
		1 Introduction
		2 Materials
			2.1 Sample Preparation
			2.2 FAK Inhibition
			2.3 Western  Blot
			2.4 Immunofluorescence Staining
			2.5 Equipment and Consumables
		3 Methods
			3.1 Preparation of Samples
				3.1.1 Surface Treatment of Patterned Molds for Soft Lithography
				3.1.2 Preparation of Patterned Polydimethylsiloxane (PDMS) Samples
				3.1.3 Preparation of Samples for Cell Culture
			3.2 Treatment of Cells with Focal Adhesion Kinase Inhibitor
			3.3 Determination of Optimal Focal Adhesion Kinase Inhibitor Concentration and Incubation  Time
				3.3.1 Concentration of pFAK Inhibitor
				3.3.2 Incubation Time with pFAK Inhibitor
			3.4 Western  Blot
				3.4.1 Sample Preparation
				3.4.2 Electrophoresis
				3.4.3 Protein Transfer
				3.4.4 Immuno-Blot
			3.5 Immunofluorescence Staining
		4 Notes
		References
Part VII: Biophysical Modeling of Cell Shape and Protein Dynamics
	Chapter 21: Long-Term Fluorescence Recovery After Photobleaching (FRAP)
		1 Introduction
		2 Materials
			2.1 Plasmids Construction
			2.2 Cell Culture and Transfection
				2.2.1 Extra Materials
			2.3 Fluorescence Recovery After Photobleaching
		3 Methods
			3.1 Sample Preparation
				3.1.1 Plasmids Construction
				3.1.2 Cell Culture
			3.2 FRAP
				3.2.1 Reaction-Advection Model for Long-Term FRAP
				3.2.2 Imaging
				3.2.3 FRAP Data Analysis
		4 Notes
		References
	Chapter 22: Simulating 3D Cell Shape with the Cellular Potts Model
		1 Introduction
		2 Materials
		3 Theory
		4 Methods
			4.1 CompuCell3D
			4.2 Morpheus
			4.3 Conclusions
		References
Index