Name: measuring the stiffness of ex vivo mouse aortas using atomic force microscopy
Uploaded: 2016-10-19T14:00:00
Description: Watch this Scientific Journal Video about Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy at JoVE.com

AFM analysis was performed on instrumentation supported by the Pennsylvania Muscle Institute and the Institute for Translational Medicine and Therapeutics, Perelman School of Medicine, the University of Pennsylvania. This work was supported by NIH grants HL62250 and AG047373. YHB was supported by post-doctoral fellowship from the American Heart Association.

Animal work in this study was approved by the Institutional Animal Care and Use Committees of the University of Pennsylvania. The methods were carried out in accordance with the approved guidelines.
1. Preparing the Mouse and Isolation of the Aorta
<ol>
	<li>Anesthetize a mouse with ketamine (80 - 100 mg/kg), xylazine (8 - 10 mg/kg) and acepromazine (1 - 2 mg/kg) intraperitoneally. Confirm anesthesia with a tail pinch test. Once the mouse is fully anesthetized, euthanize the mouse by cervica...

<ol>
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AFM indentation can be used to characterize the stiffness (elastic modulus) of cells and tissues. In this paper, we provide detailed step-by-step protocols to isolate the descending aorta and aortic arch in the mouse and determine the elastic moduli of these arterial regions ex vivo. We now summarize and discuss the technical issues and limitations of the method described in this paper.
Several technical issues can arise in the isolation and analysis of mouse aortas given their small ...

The biomechanical properties of arteries are a critical determinant in cardiovascular disease (CVD) and aging. Arterial stiffness, a major cholesterol independent risk factor and an indicator for the progression of CVD, increases with vascular injury, atherosclerosis, age, and diabetes1-8. Arterial wall stiffening is associated with increased dedifferentiation, migration, and proliferation of vascular smooth muscle cells9-12. In addition, increased arterial stiffness has been linked to enhanced macrophage adhesion1, endothelial permeability and leukocyte transmigration13, and vessel wall remodeling14,15. Thus, therapies that could prevent arterial stiffening in CVD or aging might complement currently available pharmacological interventions that treat CVD by reducing high blood cholesterol. 
AFM is a powerful analytical tool used for various physical and biological applications. AFM is increasingly used to obtain the high-resolution images and characterize the biomechanical properties of soft biological samples such as tissues and cells1,2,10,16,17 with a great degree of accuracy at nanoscale levels. A major advantage of AFM is the fact that it can be used with living cells.
This paper describes our method for measuring the elastic modulus of mouse arteries ex vivo using AFM. The described method shows how we 1) properly isolate mouse arteries (descending aorta and aortic arch) and 2) measure the elastic modulus of these tissues by AFM. Measurements of unloaded elastic moduli in arteries can help to elucidate changes in the extracellular matrix (ECM) that occur in response to vascular injury, CVD, and aging.

<table border="0" cellpadding="0" cellspacing="0" width="836">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:272px;">BioScope Catalyst AFM system</td>
			<td style="width:191px;">Bruker</td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nikon Eclipse TE 200 inverted microscope</td>
			<td>Nikon Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Silicon nitride AFM probe</td>
			<td>Novascan Technologies</td>
			<td>PT.SI02.SN.1</td>
			<td>0.06 N/m cantilever; 1 &#181;m SiO2 particle</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dumont #5 forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-10</td>
			<td>See section 1.4</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dumont #5SF forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-00</td>
			<td>See section 1.8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Fine Scissors-ToughCut</td>
			<td>Fine Science Tools</td>
			<td style="width:119px;">14058-11</td>
			<td>See section 1.4 (medium sized)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vannas-T&#252;bingen spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15008-08</td>
			<td>See section 1.6 (small sized)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">60mmTC-treated cell culture dish</td>
			<td>Corning</td>
			<td>353004</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dulbecco&#39;s Phosphate-Buffered Saline, 1X</td>
			<td>Corning</td>
			<td>21-031-CM</td>
			<td>Without calcium and magnesium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Krazy Glue instant all purpose liquid</td>
			<td>Krazy Glue</td>
			<td>KG58548R</td>
			<td>See section 2.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gel-loading tips, 1-200 &#181;L</td>
			<td>Fisher</td>
			<td>02-707-139</td>
			<td>See section 2.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tip Tweezers</td>
			<td>Electron Microscopy Sciences</td>
			<td>78092-CP</td>
			<td>See section 3.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50-mm, clear wall glass bottom dishes</td>
			<td>TED PELLA</td>
			<td>14027-20</td>
			<td>See section 4.4</td>
		</tr>
	</tbody>
</table>

measuring the stiffness of ex vivo mouse aortas using atomic force microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Figure 5A shows a phase contrast image of the descending (thoracic) aorta from a 6-month old, male C57BL/6 mouse. The AFM cantilever is in place directly above the tissue and ready for indentation. Figures 5B and 5C demonstrate representative force curves obtained by AFM indentation in contact mode. Green lines shown in Figures 5B and 5C represent the best fit curves obtained using the Hertzian model for a sphere. In Figure 5D</s...

Watch this Scientific Journal Video about Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy at JoVE.com

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

The overall goal of this protocol is to describe the procedures for using Atomic Force Microscopy to measure the Ex Vivo elastic modulus of mouse aortas. This method help answer key questions in the vascular biomechanics and disease field such as cardio vascular disease and aging. The main advantage of this technique is the AFM characterized biomechanical properties of soft biological samples such as arteries and cells with a great degree of accuracy at Danoscale levels.I'll be presenting the AFM procedures, and Doctor Shu-lin Liu, a researcher associate from our laboratory, will show how to isolate mouse aortas. To begin, euthanize a mouse and dissect it down to the aorta as described in the accompanying text protocol. Using a pair of small forceps, grasp the fat surrounding the aorta and use a small pair of scissors to carefully cut away the fat that is around the aorta.Next, gently grab the aorta with forceps and make one cut through the vessel at the beginning of the ascending aorta and another cut at the end of the descending aorta, just above the abdominal aorta. Transfer the dissected aorta to a 60 millimeter dish containing PBS without calcium or magnesium. In the dish, continue to dissect away any remaining fatty tissue from the aorta and then open the aorta longitudinally.Next, cut a 2 millimeter by 4 millimeter piece from the descending aorta, and a same size piece from the aortic arch, for analysis using the Atomic Force Microscope. Place these pieces of tissue into a fresh 60 millimeter plastic dish, and keep them moist in a drop of PBS. Carefully remove the PBS from around the tissue using a lab wipe without touching the aorta.Be certain that the luminal side of the tissue is facing up, and use five to 10 microliters of cyanoacrylate adhesive to gently glue each end of the opened aorta to the plate. It is critical that the aorta is not stretched during this process. When finished, check that the glued aorta is not folded or floating.After air drying the glue on the aorta for 30 to 60 seconds, gently add PBS and submerge the sample. Using tweezers, slide a silicone nitrate AFM probe with a spherical tip onto the probe holder. Then, firmly secure the probe holder to the ZScanner mount of the AFM head.Next, open the AFM software. Choose the experiment type by going to the Experiment Category and selecting Contact Mode. Then, go to Experiment Group and select Contact Mode in Fluid, and finally go to Experiment and select Contact Mode in Fluid.Once these are selected, click on Load Experiment. This will turn on the laser. Next prepare a sample used to align the laser as described in the accompanying text protocol.Place the sample onto the AFM. Position the laser spot onto the tip of the AFM probe, by adjusting the laser positioning knobs. Then, use the detector positioning knobs to adjust the position of the photo diode until the values for the Vertical Deflection are between zero and negative one volts, and the Horizontal Deflection is close to zero volts.Next, check that the laser Sum Signal is maximized. If necessary, readjust the position of the laser beam on the AFM tip, and the position of the photo diode, to obtain the maximum sum signal. To begin calibration, select check parameters and set the parameter for Scan Size to zero.Set the Scan Rate to one hertz. Set the Sample/Line to 256, and the Deflection Setpoint to 20 nanometers. Then select Engage.Once the probe makes contact with the surface of the plate, click on Ramp. Next, select expanded mode in the scan toolbar menu, and set the parameters as shown here. In the ramp toolbar, select ramp continuus, and obtain a force curve on the plate.In this force curve, set Channel 1 to Deflection Error in order to allow the software to graph the results as vertical deflection versus Z position. Click at the left or right ends at the force curve and drag the cursor to encompass a linear region of the force curve that will be fit with the straight line. Then, select Ramp in the toolbar and click on Update Sensitivity.Record the current value. Next, select Calibrate, and click on Detector. Input and average value from the five measurements in the Deflection Sensitivity box.Click on Withdraw two to three times, to raise the AFM head and prevent interaction between the AFM tip and the plate during thermal tune process. Then, click on Thermal Tune in the toolbar. Set Thermal Tune range to between one and 100 kilohertz, and set the Deflection Sensitivity Correction to 1.144 for V-shaped cantilevers, and 1.166 for rectangular cantilevers.In the Thermal Tune menu, click on Acquire Data to obtain a thermal tune curve. Next, place red dash to lines on either side of the curve, and select either the Lorentzian or the simple harmonic oscillator model depending on which better fits the data. Then, click on Fit Data and select Calculate Spring K to save this value.Place a 60 millimeter culture dish containing the aortic tissue onto the microscope stage, and secure the plate with the magnetic plate holder. Once the aorta is engaged with the cantilever, ensure that the piezo center is stable, and click on Ramp. Set the Ramp size to three micrometers, the Trigger mode to Relative, and the Trigger threshold to 100 nanometers.Then select ramp continuous. Observe that the point of contact between the probe and the sample occurs at approximately the center to the lower three quarters of the Z ramp cycle. If this is not observed, disengage from the sample, adjust the ramp size as needed, and reengage the sample.Next, click on Microscope in the menu bar, and select Engage Settings. Change the SPM withdraw to 30 micrometers, then click OK.After observing the force curve, click Capture in the menu bar and select Capture Filename. Enter a desired filename ending with OOO, select a designated folder, and save the data.To begin analysis, open the force curve analysis software, load the file that is to be analyzed, and modify and smooth the data as described in the accompanying text protocol. Next, select Baseline Correction in the menu bar, and set the inputs as shown here. Then, adjust the vertical dotted blue line on the graph to encompass the flat portion of the force curve and click Execute.Next, click Indentation in the toolbar menu, and set the inputs as shown here. Save these values, and analyze the Young's modulus of each of the areas of interest by measuring five force curves per area that are taken within a small distance of each other. To determine the overall stiffness of the tissue sample, up to 25 measurements should be taken over five different regions of the sample area within the tissue.These areas can be used to compare tissue in different locations, such as the aortic arch and descending aorta. In these graphs, representative force curves of the descending aorta and aortic arch are shown from the same sample of mouse aorta. Based on this data, the elastic modulus can be calculated for each group, and be used to compare between different regions or conditions.After watching this video you will have a good understanding of how to properly isolate mouse aorta and measure the elastic modulus by Atomic Force Microscopy. After attempting this procedure it's important to remember to perform the AFM measurement immediately after tissue isolation. Once mastered, the isolation of aortas can be completed in approximately 45 minutes per mouse, and the AFM measurement and analysis can be completed in one and a half hours, if it is performed properly.Following this procedure, other methods like immunoflourescence or proliferation assays can be performed in order to answer additional questions relating to vascular remodeling or vascular smooth muscle cells proliferation.

measuring-stiffness-ex-vivo-mouse-aortas-using-atomic-force

Research

JoVE Journal

Biology

Targeted Expression of GFP in the Hair Follicle Using Ex Vivo Viral Transduction

There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft during early anagen, the growth phase of the hair cycle, as well as pluripotent stem cells that play a role in hair follicle growth but have the potential to differentiate to non-follicle cells such as neurons. These properties of the hair follicle are discussed. The various cell types of the hair follicle are potential targets for gene therapy. Gene delivery system for the hair follicle using viral vectors or liposomes for gene targeting to the various cell types in the hair follicle and the results obtained are also discussed.

The hair follicle is a highly complex appendage of the skin containing a multiplicity of cell types. The follicle undergoes constant cycling through the life of the organism including growth and resorption with growth dependent on specific stem cells. The targeting of the follicle by genes and stem cells to change its properties, in particular, the nature of the hair shaft is, discussed. Hair follicle delivery systems are described, such as liposomes and viral vectors for gene therapy. The nature of the hair follicle stem cells is discussed, in particular, its pluripotency.

There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft ...

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3. On the macro scale the stiffness of tissues and organs within the human body span several orders of magnitude4. Much less is known about how stiffness varies spatially within tissues, and what the scope and spatial scale of stiffness changes are in disease processes that result in tissue remodeling. To better understand how changes in matrix stiffness contribute to cellular physiology in health and disease, measurements of tissue stiffness obtained at a spatial scale relevant to resident cells are needed. This is particularly true for the lung, a highly compliant and elastic tissue in which matrix remodeling is a prominent feature in diseases such as asthma, emphysema, hypertension and fibrosis. To characterize the local mechanical environment of lung parenchyma at a spatial scale relevant to resident cells, we have developed methods to directly measure the local elastic properties of fresh murine lung tissue using atomic force microscopy (AFM) microindentation. With appropriate choice of AFM indentor, cantilever, and indentation depth, these methods allow measurements of local tissue shear modulus in parallel with phase contrast and fluorescence imaging of the region of interest. Systematic sampling of tissue strips provides maps of tissue mechanical properties that reveal local spatial variations in shear modulus. Correlations between mechanical properties and underlying anatomical and pathological features illustrate how stiffness varies with matrix deposition in fibrosis. These methods can be extended to other soft tissues and disease processes to reveal how local tissue mechanical properties vary across space and disease progression.

The stiffness of the extracellular matrix strongly influences multiple behaviors of adherent cells. Matrix stiffness varies spatially throughout a tissue, and undergoes modification in various disease conditions. Here we develop methods to characterize spatial variations in stiffness in normal and fibrotic mouse lung tissue using atomic force microscopy microindentation.

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3.  On the macro scale the stiffness of tissues and organs ...

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3&#039;UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are strategically interposed in the path of the lymphatic vessels, allowing intimate contact of tissue antigens with all resident immune cells in the LN. Thus, understanding the cellular composition, distribution, location and interaction using ex vivo whole LN imaging will add to the knowledge on how the body coordinates local and systemic immune responses. This protocol shows an ex vivo imaging strategy following an in vivo administration of fluorescent-labeled antibodies that allows a very reproducible and easy-to-perform methodology by using conventional confocal microscopes and stock reagents. Through subcutaneous injection of antibodies, it is possible to label different cell populations in draining LNs without affecting tissue structures that can be potentially damaged by a conventional immunofluorescence microscopy technique.

This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure.

Lymph nodes (LNs) are organs spread within the body, where the innate immune responses can connect with the adaptive immunity. In fact, LNs are ...

Measuring Cell-Edge Protrusion Dynamics during Spreading using Live-Cell Microscopy

The development and homeostasis of multicellular organisms rely on coordinated regulation of cell migration. Cell migration is an essential event in the construction and regeneration of tissues, and is critical in embryonic development, immunological responses, and wound healing. Dysregulation of cell motility contributes to pathological disorders, such as chronic inflammation and cancer metastasis. Cell migration, tissue invasion, axon, and dendrite outgrowth all initiate with actin polymerization-mediated cell-edge protrusions. Here, we describe a simple, efficient, time-saving method for the imaging and quantitative analysis of cell-edge protrusion dynamics during spreading. This method measures discrete features of cell-edge membrane dynamics, such as protrusions, retractions, and ruffles, and can be used to assess how manipulations of key actin regulators impact cell-edge protrusions in diverse contexts.

This protocol aims to measure the dynamic parameters (protrusions, retractions, ruffles) of protrusions at the edge of spreading cells.

The development and homeostasis of multicellular organisms rely on coordinated regulation of cell migration. Cell migration is an essential event in the ...

Characterizing Mechanical Properties of Primary Cell Wall in Living Plant Organs Using Atomic Force Microscopy

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the plant. Many sophisticated techniques have been developed to measure these properties; however, atomic force microscopy (AFM) remains the most convenient for studying cell wall elasticity at the cellular level. One of the most important limitations of this technique has been that only superficial or isolated living cells can be studied. Here, the use of atomic force microscopy to investigate the mechanical properties of primary cell walls belonging to the internal tissues of a plant body is presented. This protocol describes measurements of the apparent Young&#39;s modulus of cell walls in roots, but the method can also be applied to other plant organs. The measurements are performed on vibratome-derived sections of plant material in a liquid cell, which allows (i) avoiding the use of plasmolyzing solutions or sample impregnation with wax or resin, (ii) making the experiments fast, and (iii) preventing dehydration of the sample. Both anticlinal and periclinal cell walls can be studied, depending on how the specimen was sectioned. Differences in the mechanical properties of different tissues can be investigated in a single section. The protocol describes the principles of study planning, issues with specimen preparation and measurements, as well as the method of selecting force-deformation curves to avoid the influence of topography on the obtained values of elastic modulus. The method is not limited by sample size but is sensitive to cell size (i.e., cells with a large lumen are difficult to examine).

Studies of cell wall biomechanics are essential for understanding plant growth and morphogenesis. The following protocol is proposed to investigate thin primary cell walls in the internal tissues of young plant organs using atomic force microscopy.

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the ...