The authors thank Drs. Scott R. Hutton and Meghan S. Blackledge for the critical review of the manuscript.&#160;This work was funded by High Point University&#8217;s Research and Sponsored Programs (MCS) and the Biotechnology Program at North Carolina State University (MCS).

1. Preparations
NOTE: The protocol described below has been optimized for the use with REF52 cells and ATM+/+ or ATM-/- human fibroblasts. Other cell types may require further optimization as described in the notes and troubleshooting sections below.
<ol>
	<li>Make 500 mL of complete cell culture medium for REF52 cells. To 500 mL of high-glucose containing Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) add 10% FBS, 2 mM L-glutamine, and 100 units/mL penicillin-streptomycin.</li>
	<li>Prepare a 25 &#956;g/mL solution of fibronectin (FN) by adding 300 &#956;L of 1 mg/mL FN solution to 12 mL of sterile 1x phosphate buffered saline (PBS), pH 7.4. Mix well.</li>
	<li>Prepare a 0.5% (w/v) delipidated (i.e., fatty acid free) bovine serum albumin (dlBSA) solution in serum free DMEM cell culture medium. Add 0.5 g of dlBSA to 100 mL of serum free DMEM medium. Mix the solution well, but do not vortex. Sterile filter the solution into a new sterile container, using a 0.22 &#956;M syringe filter before use. Store at 4 &#176;C.</li>
	<li>Make 3.7% paraformaldehyde solution by dissolving 3.7 g of paraformaldehyde in 100 mL of 1x PBS. Use gentle heat and stirring to get the paraformaldehyde into solution. 
	NOTE: The paraformaldehyde solution is light sensitive and should be protected from light. It is good for up to one week when stored at 4 &#176;C. 
	CAUTION: Paraformaldehyde is toxic, flammable, corrosive and a health hazard. Review the material safety data sheet for paraformaldehyde prior to use. Use the appropriate personal protective equipment when handling including eye shield, face shield, full-face particle respirator, gloves, and lab coat.</li>
	<li>Prepare the permeabilization solution containing 0.2% non-ionic surfactant in 1x PBS (v/v). For 100 mL, slowly add 0.2 mL of Triton X-100 to 100 mL of 1x PBS, while stirring.</li>
	<li>Make the immunofluorescence blocking buffer containing 2.5% BSA, 5% goat serum, and 0.05% non-ionic surfactant (w/v/v) dissolved in 1x PBS solution. For 100 mL, add 5 mL of goat serum, 2.5 g of BSA and 0.05 mL of Triton X-100 in ~ 95 mL 1x PBS, while stirring.</li>
	<li>Grow REF52 cells in DMEM complete culture medium in a 10 cm2 (or any other vessel size) cell culture-treated plate in a cell culture incubator at 37 &#176;C and 5% CO2.</li>
</ol>
2. Coating cell culture plates with the extracellular matrix protein fibronectin
NOTE: Perform this section using aseptic technique and sterile reagents in a BSL-2 certified laminar flow hood. Refer to Figure 1A for an overview of key steps prior to beginning.
<ol>
	<li>Using a tissue culture certified 24-well plate, place one glass coverslip (12-Cir-1) in each well. Label the plate according to Figure 1B.</li>
	<li>Pipette 500 &#956;L of the 25 &#956;g/mL FN solution to each well of a 24-well plate.</li>
	<li>Pipette the solution over each coverslip a few times to ensure even coating and complete submersion. Place the lid back on the plate.</li>
	<li>Incubate the plate in a cell culture incubator at 37 &#176;C and 5% CO2 for 1 h. 
	NOTE: Alternatively, incubate overnight at 4 &#176;C.</li>
	<li>After 1 h, remove the plate from the incubator and aspirate the FN solution from the wells.</li>
	<li>Wash wells three times with 500 &#956;L of 1x PBS. Aspirate the final wash of 1x PBS.</li>
	<li>Block wells with 500 &#956;L of 0.5% dlBSA solution for a minimum of 15 min at 37 &#176;C and 5% CO2.</li>
	<li>Aspirate the dlBSA solution prior to plating cells in step 3 below. 
	NOTE: If storing plates, add 500 &#956;L of 1x PBS to each coverslip after aspiration of the dlBSA solution. Plates can then be kept at 4 &#176;C for up to one week.</li>
</ol>
3. Preparing anchorage-dependent cells for the adhesion assay
NOTE: Perform this section using aseptic technique and sterile reagents in a BSL-2 certified laminar flow hood.
<ol>
	<li>At least 30 min prior to the cell plating, pre-warm the following solutions: DMEM complete medium, dlBSA solution, 1x PBS, 0.5% trypsin-EDTA solution, and trypsin neutralizing serum (TNS) in a 37&#176;C water bath.</li>
	<li>Starting with a confluent monolayer of REF52 cells in a 10 cm2 dish, wash cells twice with 6 mL of warm 1x PBS. Serum starve the cells for at least 1 h (depending on cell type) in 6 mL of warm dlBSA solution at 37 &#176;C and 5% CO2.</li>
	<li>Wash cells with 6 mL of warmed 1x PBS, aspirate PBS and add 1.5 mL of warm 0.5% Trypsin-EDTA solution.</li>
	<li>Place cells in a cell culture incubator at 37 &#176;C and 5% CO2 for ~2 min.</li>
	<li>Observe cells under a light microscope to ensure the detachment is complete. If cells are still adherent after tapping the plate on the bench top, return to the 37 &#176;C incubator for an additional 2 min. Trypsinize the cells for as little time as is necessary.</li>
	<li>Pipette 1.5 mL of warm trypsin neutralizing solution (TNS) to the dish to stop trypsinization and collect detached cells. Pipette the solution up and down over the bottom of the plate numerous times to remove all remaining adherent cells. If cells appear clumpy, further triturate the cell suspension by gently pipetting up and down over the back of the dish.</li>
	<li>Count cells using trypan blue exclusion and a hemocytometer under a light microscope. Alternatively, use an automated cell counter.</li>
	<li>Remove an appropriate amount of cells to create a 1.0 - 3.0 x 104 cells/mL cell suspension in 5 mL of dlBSA in a 15 mL conical tube.</li>
	<li>Centrifuge cells at ~ 300 x g for 5 min using a fixed angle rotor in a table-top centrifuge.</li>
	<li>Aspirate the supernatant from the cell pellet, and resuspend cells in a total of 7 mL of warm dlBSA solution. Do not allow the cells to be overly confluent upon coverslip plating, but evenly distributed with few cells touching one another.</li>
	<li>Evenly divide the cell suspension into two 15 mL conical tubes, one for the vehicle alone control (DMSO) and one for Ku55933 (ATM kinase inhibitor, oxidant)5. Ensure each tube contains 3.5 mL of the cell suspension.</li>
	<li>Using a tube rotator, revolve the tubes at 37 &#176;C for 90-120 min in a cell culture incubator.</li>
	<li>30 min before plating, add a final concentration of 10 &#956;M Ku55933 and DMSO (1:1,000) to each respective tube. Place the cell suspension back on the rotator for the remaining time.</li>
	<li>Immediately prior to plating the cells, retrieve the 24-well plate from the incubator and aspirate the dlBSA solution.</li>
	<li>After revolving the cell suspension for 90-120 min, remove 500 &#956;L of cell suspension from each treatment group and add to one FN coated coverslip in the 24-well plate from step 2 as illustrated (Figure 1B). Return the plate to the 37 &#176;C and 5% CO2 cell culture incubator and the cell suspension back to the rotation.</li>
	<li>After plating the cell suspension on the FN covered-coverslips, allow cells to adhere for the desired length of time (e.g., 10 min, 15 min, 20 min, 30 min) and then immediately proceed to step 4.</li>
</ol>
4. Cell fixation and antibody staining for immunofluorescence
NOTE: The following steps are performed under non-sterile conditions and at room temperature unless otherwise stated.
<ol>
	<li>After the desired time for adhesion has passed, aspirate the cell solution from each coverslip in the plate.</li>
	<li>Using the sides of the well, gently dispense 500 &#956;L of 3.7% paraformaldehyde solution onto each coverslip and wait 10-15 min.</li>
	<li>Remove the paraformaldehyde solution and wash each coverslip with 500 &#956;L of 1x PBS for a total of two times. 
	NOTE: Dispose of paraformaldehyde waste responsibly, according to the institution&#8217;s environmental health and safety plan.</li>
	<li>Aspirate the PBS, and permeabilize cells on each coverslip with 500 &#956;L of 0.2% Triton X-100 in 1x PBS (v/v) for 10-15 min at room temperature.</li>
	<li>Wash each coverslip with 500 &#956;L of 1x PBS three times.</li>
	<li>Block cells on each coverslip with 500 &#956;L of immunofluorescence blocking buffer containing 5% goat serum, 2.5% BSA and 0.05% Triton X-100 dissolved in a 1 x PBS solution for 30-60 minutes.</li>
	<li>Dilute the primary anti-paxillin antibody (1:250) in the blocking buffer. Mix well and add 200 &#956;L of the antibody solution to each coverslip. Incubate at room temperature for at least 1 h. 
	NOTE: Alternatively, the primary antibody solution can be incubated overnight at 4 &#176;C. There are many common focal adhesion markers that could be used for staining adhesion complexes and subsequent FA analysis. These include antibodies against the following proteins: integrin subunits (&#946;1, &#945;5, or &#945;V), talin, or vinculin2.</li>
	<li>Aspirate the antibody solution, and wash each coverslip with 500 &#956;L of 1x PBS three times for 10 min each. Protect the samples from light from this point forward.</li>
	<li>Dilute the phalloidin F-actin probe conjugated to the red fluorescent Alexa 594 dye (1:1000) and goat-anti mouse 488 fluorescent secondary antibody (1:400) in the same blocking buffer solution. Mix well and add 200 &#956;L of the antibody solution to each coverslip for 30 min. 
	NOTE: Fluorescently conjugated secondary antibodies from other species may be used as well. However, the use of antibodies from other species will require modification of the blocking buffer serum.</li>
	<li>Aspirate the antibody solution, and wash each coverslip with 500 &#956;L of 1x PBS three times for 10 min each.</li>
	<li>Aspirate the 1x PBS and rinse one time with 500 &#956;L of dIH2O.</li>
	<li>Mount coverslips onto microscope slides using anti-fade mounting medium containing DAPI.</li>
	<li>Leave microscope slides to set overnight in the dark at room temperature.</li>
	<li>Store microscope slides in the dark at 4 &#730;C for the long-term storage and until imaging. 
	NOTE: Image using standard immunofluorescence techniques. It is recommended to use a high-powered oil immersion 60x objective lens to ensure enough resolution to note the focal adhesions and peripheral ruffles at cell edges. Acquire images of 20-30 cells in multiple fields of view for each coverslip under each treatment condition and time. From combined replicates, this should yield at least 60 cells in order to perform statistical analysis. Save and export fluorescence images as a .TIFF file with a minimum of 300 dpi resolution.</li>
</ol>
5. Quantifying stress fibers, cell circularity, and focal adhesion formation
NOTE: The following image analyses are performed using the latest version of the open source imaging processing package Fiji Is Just Image J (Fiji), which can be downloaded free of charge at (http://fiji.sc/).
<ol>
	<li>General image processing 
	NOTE: All images will need to be prepared for computational analyses by performing steps 5.1.1-5.1.5 below (Figure 2). Afterward, any or all subsequent quantification procedures may be selected.
	<ol>
		<li>Open the .TIFF fluorescence image using Fiji. Ensure the images are 8-bit and grayscale.</li>
		<li>Select Image-Adjust-Window/level and select Auto (Figure 2A).</li>
		<li>Select Process-Subtract Background to subtract the background fluorescence. Check Sliding Paraboloid and select the option of a Rolling Ball Radius of 50 pixels (Figure 2B). 
		NOTE: To verify the proper size for the rolling ball radius, select the Line Tool and draw a radius on the largest adhesion in the image. Select Measure to verify the length of the line drawn. If the value of the radius is too large, features including adhesions will be lost in the image. If the radius is too small, it will give rise to artifacts in the processed image due to background noise.</li>
		<li>Select Image-Adjust- Brightness/Contrast to check the intensity of the adhesion over the background. Adjust if necessary. 
		NOTE: To optimize the brightness/contrast and avoid saturating the signal, use the lookup tool of the image to examine its histogram to adjust the brightness/contrast.</li>
		<li>Select the following parameters under Analyze-Set Measurements: Area, Mean Gray Value, Shape Descriptors, and Integrated Density.</li>
	</ol>
	</li>
	<li>Stress fiber formation analysis 
	NOTE: Stress fibers can be quantified multiple ways depending on the phenotype.
	<ol>
		<li>Count the number of cells with stress fibers as a percentage over the total number of cells. This analysis is best if there are visual differences in the number of stress fibers formed under different experimental conditions.</li>
		<li>Count the number of stress fibers that transverse the cell. This analysis allows for the comparison of the number of stress fibers formed per cell.</li>
		<li>Measure the total fluorescence intensity given by the phalloidin (e.g., F-actin) staining per cell17,18. This method will highlight drastic increases/decreases in fluorescence intensity due to F-actin staining.
		<ol>
			<li>Set the measurement parameters in step 5.1.5 above.</li>
			<li>Select the Freehand Tool in the Fiji toolbar and manually trace the cell(s) of interest. Select Analyze-Measure. A new window will appear showing the selected measurement parameters.</li>
			<li>Select the Freehand Tool in the Fiji toolbar and manually trace an empty space with no cells present. Select Analyze-Measure. This measurement will serve as the background fluorescence.</li>
			<li>Use the equation below to determine the total F-actin fluorescence per cell: 
			<img alt="Equation 1" src="/files/ftp_upload/59989/59989equ01.jpg" /> 
			NOTE: The resulting measurement can be normalized and compared to other cells to give F-actin fluorescence per cell.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Cell circularity analysis 
	NOTE: Information on cell area (an indicator of cell spreading over time), as well as, the circularity can also be recorded. This measurement is given as a ratio between 0 to 1 as a way to quantify cells that are elongated to round, respectively.
	<ol>
		<li>Select the Freehand Tool in the Fiji toolbar, and trace an individual cell. Select Image-Measure and record the cell area and perimeter measurements for each cell. Repeat this procedure for each cell. 
		NOTE: Under the Set Measurements function, circularity is provided as the Shape Descriptors measurement (step 5.1.5).</li>
		<li>Manually count actin-enriched ruffling or protrusions per cell as depicted in Figure 3 and Figure 4.</li>
	</ol>
	</li>
	<li>Focal adhesion analysis 
	NOTE: Before performing focal adhesion analysis, install the Mexican Hat Filter plugin on the latest version of Fiji. The following protocol has been modified from previous studies19,20,21.
	<ol>
		<li>Select Process-Enhance Local Contrast (Clahe) using a block size of 19, histogram bins 256, and a maximum slope of 6, with no mask and not fast. (Figure 2C)</li>
		<li>Select Process-Filters- Gaussian Blur with a Sigma (Radius) of 2.0 to filter the image (Figure 2D).</li>
		<li>Select Plugins-Mexican Hat Filter (Mhf) with a Radius of 2.0 (Figure 2E).</li>
		<li>Run Threshold and select Dark Background and Over/Under using either Huang or Isodata as the thresholding method. Select Auto-Threshold. 
		NOTE: This step ensures that adhesions are highlighted, but also distinct from one another.</li>
		<li>Select Analyze-Analyze Particles with the following parameters selected: size=20, pixels-infinity and circularity=0.00-0.99. Check the outlines to ensure the proper detection and separation of focal adhesions. 
		NOTE: These results yield the number, area, and shape description of individual focal adhesions.</li>
	</ol>
	</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+cellular+adhesion+to+extracellular+matrix+components+by+single-cell+force+spectroscopy.">Quantifying cellular adhesion to extracellular matrix components by single-cell force spectroscopy.</a> Nature Protocols. 5 (7), 1353-1361 (2010).</li><li>Brown, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+mild+trypsinization+conditions+in+the+detachment+of+endothelial+cells+to+promote+subsequent+endothelialization+on+synthetic+surfaces.">The use of mild trypsinization conditions in the detachment of endothelial cells to promote subsequent endothelialization on synthetic surfaces.</a> Biomaterials. 28 (27), 3928-3935 (2007).</li></ol>

The authors declare that they have no competing financial interests.

The protocol described here is a versatile and economical way to rapidly screen a number of anchorage-dependent cell types for dynamic cytoskeleton remodeling during cell spreading. In particular, this method quantitatively examines stress fiber and focal adhesion formation during oxidative stress when cells adhere to FN (Figure 1A). Moreover, these cellular phenotypes may suggest a regulatory role for members of the Rho family of small GTPases since they have documented roles during cell attachment and spreading15,16,22. However, additional biochemical techniques would be required to identify the possible involvement of GTP-bound, active Rho family proteins.
The presented protocol utilizes immunofluorescence detection of F-actin and paxillin to specifically examine cell adhesion and spreading of immortalized fibroblast cells on FN after oxidative stress induced by ATM kinase inhibition (Figure 2, Figure 3 and Figure 4). However, this procedure can also be adapted for use on other ECM proteins and/or for other adherent cell types. When adapting to other cell lines, it is important to optimize the experimental conditions, particularly: cell number/density, time of serum starvation, ECM protein concentration, and oxidative stress treatment conditions. When testing the effects of an unknown stimulus on adhesion and spreading dynamics, it is necessary to include both negative and positive control samples to verify that the assay is functioning correctly. Negative control samples can include an untreated or vehicle-only sample, while a positive control should induce oxidative stress (e.g., H2O2). Furthermore, although not discussed here, it is also essential to utilize the proper antibody controls. It is recommended that three separate controls be used for each antibody to verify its specificity and to identify any potential fluorescence bleed-through23,24. These include: 1) a primary antibody control to ensure specific binding of the primary antibody to the antigen and to confirm that antigen binding occurs under the fixation conditions used, 2) a secondary antibody control (for non-secondary conjugated-antibodies) that shows specificity to the primary antibody, and 3) fluorophore controls that ensure the fluorophore added is not the result of endogenous fluorescence or bleed-through from another antibody.
While this assay is useful for analyzing the early kinetic events of adhesion assembly and spreading, it is not suitable for the detailed examination of adhesion disassembly or adhesion strength and cellular reinforcement. The latter requires the use of long-term imaging bio-stations or single-cell force spectroscopy techniques. The latter techniques include atomic-force microscopy, optical tweezers, tension sensors of adhesion proteins such as vinculin25 or talin26,27, and 3D-force microscopy28.
Critical steps in the protocol include thoroughly coating coverslips with FN. This is necessary for the uniform spreading and adhesion of cells. It is therefore important to pipette the FN solution up and down over the coverslips multiple times prior to incubation. Coverslips must remain fully submerged in the FN solution during the incubation time. FN coated coverslips can be stored for up to 2 weeks at 4 &#176;C.
Cell density is also important, as cells that are plated too densely will not achieve maximum spreading circumference. Furthermore, it would not be possible to distinguish individual focal adhesions or cellular ruffles for each individual cell. It is, therefore, necessary to count the cells using a hemocytometer or automated cell counter prior to placing the cells in suspension. While an estimate of cell density is provided for REF52 cells, this will need to be empirically determined for other cells under study. Cells should be plated sparsely enough that few cells overlap, allowing them to spread fully (Figure 5).
Other critical steps in the protocol to consider are fluorescently conjugated phalloidin-Alexa 594 F-actin probe and secondary antibodies are light sensitive. Therefore, samples should be minimally exposed to light after the application of these reagents. Furthermore, a number of agents that induce oxidative stress have short half-lives. It is, therefore, necessary to test the chosen oxidant for optimal dose and exposure time to achieve peak activity.
The following sections contain trouble-shooting tips with regards to FN concentration, serum starvation conditions, and cell detachment methodologies. These tips are useful when adapting the protocol for other cells lines and/or treatment conditions.
For consistent FA analysis, optimal attachment and spreading conditions are necessary, which will vary depending upon the ECM ligand. For FN, begin using a dynamic range of 10-30 &#956;g/mL. At higher concentrations of FN, there is little difference in cell attachment. However, some cell types do not efficiently spread at higher concentrations of ECM.
Each cell type responds differently to conditions of serum starvation. REF52 cells can easily be starved overnight without any loss of viability, however, this is not true for all cell lines. Therefore, it is necessary to determine the degree to which the cell line under study can withstand serum starvation.
For spreading assays, cells need to be trypsinized for as little time as possible for reproducible results29. Following cell detachment by trypsinization, cell surface receptors, their cognate ligands, and Rho GTPase activity require a recovery period to return to steady-state levels14,15. The trypsinization procedure outlined in this protocol should enable cells to retain most of their cell surface FN-binding receptors29. However, certain cell types may require alternative methods of cell detachment to completely prevent digestion of cell surface receptors. These methods include chelating cells using EDTA-based solutions (e.g., Versene) or milder enzymatic dissociation solutions (e.g., Accutase). Use of these dissociation solutions may leave cell-cell adhesions intact resulting in cell clumps. It is therefore important to completely resuspend individual cells to ensure experimental reproducibility.

Cell-matrix adhesions (i.e., focal adhesions) are large and dynamic multimolecular protein complexes which mediate cell adhesion and spreading. These processes are critical for tissue development, maintenance, and physiological function. Focal adhesions are composed of membrane-bound receptors, such as integrins, as well as scaffolding proteins that link cytoskeletal actin to the extracellular matrix (ECM)1. These complexes are capable of responding to physiochemical cues present in the extracellular environment through the activation of various signaling transduction pathways. As such, focal adhesions serve as signaling centers to propagate extracellular mechanical cues into a number of cellular processes including directed migration, cell cycle regulation, differentiation, and survival1,2. One group of signaling molecules that regulate and interact with focal adhesions includes members of the Rho family of small GTPases. Rho GTPases are key proteins that regulate cell migration and adhesion dynamics through their specific spatiotemporal activation3. Not surprisingly, dysregulation of Rho protein function has been implicated in a number of human pathologies such as metastasis, angiogenesis, and others. Of particular interest, cellular redox status plays a predominant role in the modulation of cell migration and adhesion. Alterations in redox homeostasis, such as increases in reactive oxygen species (ROS), have been demonstrated to regulate Rho protein activity, as well as adhesion, in a number of cell types and human diseases4,5,6,7,8. For example, individuals suffering from the neurological disorder ataxia-telangiectasia (A-T), which is caused by a mutation in the DNA damage repair serine/threonine kinase A-T-mutated (ATM), have an increased risk of metastatic cancer9,10. Loss of ATM kinase activity in these patients and cell lines, either through genetic mutation or chemical inhibition, results in high levels of oxidative stress due to dysfunction of the pentose phosphate pathway7,11,12. Moreover, recent studies from the laboratory have highlighted a pathophysiological role for ROS in A-T by altering cytoskeletal dynamics (i.e. adhesion and spreading) as a direct result of activating Rho family GTPases in vitro5. Ultimately, these alterations in cytoskeletal dynamics caused by Rho family activation may lead to the increased risk of metastatic cancer noted in A-T patients5,13. Therefore, understanding the interplay between cell-matrix interactions during oxidative stress can provide insights into the regulation of adhesion and spreading. These studies can also set the stage for further investigations into a possible role for Rho family GTPases in these signaling processes.
Described herein is a protocol to study the early cellular dynamics of adhesion assembly and spreading during oxidative stress caused by inhibition of ATM kinase activity. This assay is based on the well-characterized mechanism of anchorage-dependent cells adhesion to the ECM protein fibronectin (FN). When cells maintained in suspension are plated onto FN, several Rho GTPases coordinate the control of the actin cytoskeletal remodeling3,14. Morphological changes are observed as cells shift from round and circular in appearance to flattened and expanded. Concomitant with these observations is the development of numerous matrix adhesions with the ECM. These changes are attributed to the biphasic activation of RhoA with Rac1 during the first hour as cells adhere and spread 15,16.
A variety of methods have been utilized to examine adhesion morphology and dynamics as well as cell spreading. However, these methods rely on sophisticated long-term, live-imaging total internal reflection fluorescence (TIRF) or confocal microscopy systems. Thus, users must have access to specialized equipment and software. Furthermore, the set-up time required by these bio-imaging systems makes capturing early adhesion events challenging, especially when testing multiple inhibitors or treatment conditions concurrently.
The methods detailed, herein, provide a straightforward, economical, yet quantitative way to assess parameters that govern the adhesion assembly and spreading in vitro. The protocol is performed using commonly available laboratory equipment, such as an epifluorescence microscope and CCD camera. This assay involves applying anchorage-dependent cells to an FN-coated surface after a period of oxidative stress caused by chemical inhibition of ATM kinase activity, which has been demonstrated previously5. Following plating, cells are allowed to attach and adhere for specified lengths of time. Unattached cells are washed away, while attached cells are fixed and labeled with fluorescence-based antibodies to markers of adhesion (e.g., paxillin) and spreading (e.g., F-actin)2,5. These proteins are then visualized and recorded using an epifluorescence microscope. Subsequent data analysis is performed using freely available Fiji software. Moreover, this method can be adapted to examine adhesion dynamics under a wide range of conditions including different ECM proteins, treatment with various oxidants/cell culture conditions or a variety of anchorage-dependent cell lines to address a broad range of biological questions.

<table><tbody><tr><td>0.05% Trypsin-EDTA (1x)</td><td>Gibco by Life Technologies</td><td>25300-054</td><td>cell dissociation</td></tr><tr><td>10 cm2 dishes</td><td>Cell Treat</td><td>229620</td><td>sterile, tissue culture treated</td></tr><tr><td>15 mL conical tubes</td><td>Fisher Scientific</td><td>05-539-5</td><td>sterile</td></tr><tr><td>1X Phosphate Buffered Saline</td><td>Corning Cellgro</td><td>21-031-CV</td><td>PBS, sterile, free of Mg2+ and Ca2+</td></tr><tr><td>24-well cell culture treated plates</td><td>Fisher Scientific</td><td>07-200-740</td><td>sterile, tissue culture treated</td></tr><tr><td>4&amp;deg;C refrigerator</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Mouse IgG anti-paxillin primary antibody (clone 165)</td><td>BD Transduction Laboratories</td><td>610620</td><td>marker of focal adhesions</td></tr><tr><td>Aspirator</td><td>Argos</td><td>EV310</td><td></td></tr><tr><td>Biosafety cabinet</td><td>Nuair</td><td>NU-477-400</td><td>Class II, Type A, series 5</td></tr><tr><td>Delipidated Bovine Serum Albumin (Fatty Acid Free) Powder</td><td>Fisher Scientific</td><td>BP9704-100</td><td>dlBSA</td></tr><tr><td>Dimethyl Sulfoxide</td><td>Fisher Scientific</td><td>BP231-100</td><td>organic solvent to dissolve Ku55933</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Media, High Glucose</td><td>Fisher Scientific</td><td>11965092</td><td>REF52 base cell culture medium</td></tr><tr><td>Fetal bovine serum</td><td>Fisher Scientific</td><td>16000044</td><td>certified, cell culture medium supplement</td></tr><tr><td>Fiji</td><td>National Institutes of Health</td><td>http://fiji.sc/</td><td>image analysis program</td></tr><tr><td>Filter syringe</td><td>Fisher Scientific</td><td>6900-2502</td><td>0.2 &amp;micro;M, sterile</td></tr><tr><td>Glass coverslips (12-Cir-1.5)</td><td>Fisher Scientific</td><td>12-545-81</td><td>autoclave in foil to sterilize</td></tr><tr><td>Goat anti-mouse IgG secondary antibody Alexa Fluor 488</td><td>Invitrogen</td><td>A11001</td><td>fluorescent secondary antibody, light sensitive</td></tr><tr><td>Goat Serum</td><td>Gibco by Life Technologies</td><td>16210-064</td><td>component of blocking solution for immunofluorescence</td></tr><tr><td>Hemocytometer</td><td>Fisher Scientific</td><td>22-600-107</td><td>for cell counting</td></tr><tr><td>Human Plasma Fibronectin</td><td>Gibco by Life Technologies</td><td>33016-015</td><td>FN</td></tr><tr><td>IX73 Fluorescence Inverted Microscope</td><td>Olympus</td><td></td><td>microscope to visualize fluorescence, cell morphology, counting and dissociation</td></tr><tr><td>Ku55933</td><td>Sigma-Aldrich</td><td>SML1109-25MG</td><td>ATM kinase inhibitor, inducer of reactive oxygen species</td></tr><tr><td>L-glutamine</td><td>Fisher Scientific</td><td>25-030-081</td><td>cell culture medium supplement</td></tr><tr><td>Monochrome CMOS 16 bit camera</td><td>Optimos</td><td></td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148-500G</td><td>PFA, fixative for immunofluorescence</td></tr><tr><td>Penicillin-streptomycin</td><td>Fisher Scientific</td><td>15-140-122</td><td>P/S, antibiotic solution for culture medium</td></tr><tr><td>Alexa Fluor 594 phalloidin (F-actin probe)</td><td>Invitrogen</td><td>A12381</td><td>marker of F-actin, light sensitive</td></tr><tr><td>ProLong Gold Anti-fade reagent with DAPI</td><td>Invitrogen</td><td>P36941</td><td>cover slip mounting media including nuclear dye DAPI, light sensitive</td></tr><tr><td>REF52 cells</td><td></td><td></td><td>Graham, D.M. et. al. Journal of Cell Biology 2018</td></tr><tr><td>Stir plate with heat control</td><td>Corning Incorporated</td><td>PC-420D</td><td></td></tr><tr><td>Syringe</td><td>BD Biosciences</td><td>309653</td><td>60 mL syringe</td></tr><tr><td>Tissue culture incubator</td><td>Nuair</td><td></td><td></td></tr><tr><td>Triton X-100</td><td>Fisher Scientific</td><td>BP151-500</td><td>detergent used to permeabilize cell membranes</td></tr><tr><td>Trypan Blue Solution</td><td>Fisher Scientific</td><td>15-250-061</td><td>for cell counting</td></tr><tr><td>Trypsin Neutralizing Solution (1x)</td><td>Gibco by Life Technologies</td><td>R-002-100</td><td>TNS, neutralizes trypsin instead of fetal bovine serum</td></tr><tr><td>tube rotator</td><td>Fisher Scientific</td><td>11-676-341</td><td></td></tr><tr><td>water bath</td><td>Fisher Scientific</td><td>FSGPD02</td><td></td></tr></tbody></table>

examining the dynamics of cellular adhesion and spreading of epithelial cells on fibronectin during oxidative stress

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the homeostasis of adult tissues. Interestingly, oxidative stress can alter these processes, thus contributing to the pathophysiology of diseases such as metastatic cancer. Therefore, understanding the mechanism(s) of how cells attach and spread on the ECM during perturbations in redox status can provide insight into normal and disease states. Described below is a step-wise protocol that utilizes an immunofluorescence-based assay to specifically quantify cell adhesion and spreading of immortalized fibroblast cells on fibronectin (FN) in vitro. Briefly, anchorage-dependent cells are held in suspension and exposed to the ATM kinase inhibitor Ku55933 to induce oxidative stress. Cells are then plated on FN-coated surface and allowed to attach for predetermined periods of time. Cells that remain attached are fixed and labeled with fluorescence-based antibody markers of adhesion (e.g., paxillin) and spreading (e.g., F-actin). Data acquisition and analysis are performed using commonly available laboratory equipment, including an epifluorescence microscope and freely available Fiji software. This procedure is highly versatile and can be modified for a variety of cell lines, ECM proteins, or inhibitors in order to examine a broad range of biological questions.

A general schema of the experimental set-up
Figure 1 represents the general schema for the cell adhesion and spreading protocol beginning with serum starvation of REF52 cells and ending with computational analysis of acquired fluorescence images. Key steps in the protocol are illustrated in the timeline. Of note, step 2 of the protocol describes the preparation of the FN-coated coverslips, which should be performed concurrently with step 3: serum starving REF52 cells prior to placing them in suspension (Figure 1A). An example of a mock-labeled 24-well plate indicating treatment groups and duration of cell adhesion prior to fixation of samples for fluorescence microscopy (Figure 1B).
Immunofluorescence image processing for focal adhesion quantification
REF52 cells were held in suspension for 90 minutes, plated on FN, and allowed to adhere for an additional 15 minutes. After fixation and staining with an anti-paxillin antibody, fluorescent 8-bit grayscale images of the cells were acquired. Image processing analysis was performed according to the protocol delineated in Step 5. Shown are representative images of each distinct processing step including the original image (Figure 2A), and images following background subtraction (post-Rolling Ball) (Figure 2B), CLAHE (Figure 2C), Gaussian Blur (Figure 2D) and Mexican Hat Filter (Post-MHF) (Figure 2E) filtering steps. After completing all image processing steps, individual focal adhesions should be prominent, in focus, and readily distinguishable from one another (Figure 2E). After the images are filtered, the focal adhesions can be quantified and their area measured (steps 5.1 and 5.4).
Visualization of cell adhesion and spreading on FN after oxidative stress
A representative grayscale fluorescence image of anti-paxillin (focal adhesion marker) (Figure 3, top panel) and phalloidin F-actin probe staining (Figure 3, bottom panel) of REF52 cells after plating on FN with or without Ku55933 (ROS-inducing agent) treatment. Prior to the assay, REF52 cells were serum starved for 1 h. Following serum starvation, cells were held in suspension while being treated with either vehicle alone or 10 &#956;M Ku55933 to induce oxidative stress. Cells were plated on FN-coated coverslips for the indicated times, fixed, and then stained with an antibody to focal adhesions and phalloidin to detect F-actin proteins. Prominent focal adhesions and stress fibers should be readily visible in REF52 cells after being allowed to adhere for 20-30 min on FN. Plated cells should not overlap with one another to permit full cellular spreading after adhesion. Notice the clear, distinct cell edges as well as space for individual cells to spread (Figure 3). F-actin enriched ruffles at the leading edge of cell membranes are visible and indicated with an arrow (Figure 3, bottom panel).
Graphical representation of quantified fluorescence images of stress fibers and the degree of cell spreading
Examples of quantified images displayed in bar graph form representing the percentage of cells with stress fibers and the degree of cell spreading with and without Ku55933 treatment at various times after adhesion. Fluorescent images of phalloidin F-actin probe and anti-paxillin staining, similar to images shown in Figure 3, were analyzed for the percentage of stress fibers and cell spreading (i.e., circularity index) using the image analysis procedures described in step 5 of the protocol. Notably, oxidant treatment caused a significant increase in stress fiber formation at all adhesion time points examined (Figure 4A) and a decrease in cell spreading following 15 minutes of cell adhesion to FN (Figure 4B).
Non-quantifiable immunofluorescence images due to cellular over confluency
Serum-starved REF52 cells were held in suspension for 90 minutes, during which time they were treated with 10 &#956;M Ku55933 to induce ROS formation. Cells were then plated on FN and allowed to adhere for 20 minutes, after which they were fixed and stained with anti-paxillin or phalloidin-Alexa 594 F-actin probe. Plating at higher cell densities leads to cellular crowding, which prohibits cells from fully spreading due to over confluency. Notice cell edges are indistinguishable from adjacent cells (yellow arrows) (Figure 5A). As a result, quantification of individual cells is precluded, and spreading circumference cannot be accurately determined. In Figure 5B, a separate cell line, mouse embryonic fibroblasts (MEF), were held in suspension and then plated on FN for 30 minutes. Cells were then fixed and stained with an anti-paxillin antibody. Out of focus cells are denoted by red arrows (Figure 5B). Furthermore, the cross-reactivity of the anti-paxillin antibody with cellular debris (blue arrow) will alter thresholding during quantitative image analysis (Part 5) and should not be included in the analysis (Figure 5B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59989/59989fig1d.jpg" /> 
Figure 1: Time-line of protocol and example 24-well plate set-up. 
(A) The time-line highlights key steps in the cell adhesion and spreading procedure. (B) Representative labeled 24-plate, illustrating treatment groups and times for cell adhesion. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59989/59989fig1alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59989/59989fig02.jpg" /> 
Figure 2: Examples of representative immunofluorescence images following image-processing. 
REF52 cells were held in suspension for 90 min, plated on FN, and allowed to adhere for 15 min. Cells were fixed and stained with an anti-paxillin antibody. (A) Original image and images following (B) Background subtraction (post-Rolling Ball), (C) CLAHE, (D) Gaussian Blur and (E) Mexican Hat Filter (Post-MHF) filtering steps. Bar, 20 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59989/59989fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59989/59989fig3a.jpg" /> 
Figure 3: Representative immunofluorescence images of anti-paxillin and phalloidin F-actin probe stained REF52 cells plated on FN. 
Prior to the assay, REF52 cells were serum starved for 1 h. Following serum starvation, cells were held in suspension while treated with either vehicle alone or 10 &#956;M Ku55933 to cause oxidative stress. Cells were plated on FN-coated coverslips for the indicated times, fixed and stained with an antibody to focal adhesions and phalloidin to detect F-actin proteins. F-actin enriched ruffles at the leading edge of cell membranes are indicated with an arrow. Bar, 40 &#956;m. This figure has been modified from Tolbert et al.5 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59989/59989fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59989/59989fig4a.jpg" /> 
Figure 4: Quantification of immunofluorescence images. 
Graphs illustrating (A) the percentage of cells exhibiting stress fibers and (B) cell circularity measurements. Cell circularity was defined as the cell area divided by the cell perimeter. Values ranged from 0-1.0 indicating an elongated or rounded morphology, respectively. Error bars indicate S.E.M. Student&#8217;s t-test for paired samples *p&#60;0.01 from experiments performed in triplicate. This figure has been modified from Tolbert et al.5 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59989/59989fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59989/59989fig5a.jpg" /> 
Figure 5: Non-quantifiable immunofluorescence images. 
(A) Serum-starved REF52 cells were held in suspension for 90 min, while treated with 10 &#956;M Ku55933. Cells were plated on FN and allowed to adhere for 20 min. Cells were fixed and stained with anti-paxillin or phalloidin-Alexa 594 F-actin probe. Cell edges are shown by yellow arrows. (B) MEF cells were held in suspension and then plated on FN for 30 min. Cells were fixed and stained with an anti-paxillin antibody. Out of focus cells are denoted by red arrows and cross-reactivity of anti-paxillin antibody with cellular debris are denoted with blue arrows. Bar, 30 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59989/59989fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress at JoVE.com

Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress

This method is useful for quantifying the early dynamics of cellular adhesion and spreading of anchorage-dependent cells onto the fibronectin. Furthermore, this assay can be used to investigate the effects of altered redox homeostasis on cell spreading and/or cell adhesion-related intracellular signaling pathways.

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the ...

examining-dynamics-cellular-adhesion-spreading-epithelial-cells-on

Nanyang Technological University

Research

JoVE Journal

Biochemistry

7.4K Views.  University of North Carolina at Chapel Hill. Deze methode zal onderzoekers in staat stellen om de vroege dynamiek van cellulaire hechting en verspreiding van verankering-afhankelijke cellen te kwantificeren op de extracellulaire matrix eiwit fibronectin tijdens oxidatieve stress. Deze techniek is veelzijdig en kan worden aangepast om de cytoskeletale dynamiek onder een breed scala aan omstandigheden te onderzoeken. Bovendien worden gegevensverwerving en -analyse uitgevoerd met behulp van gemeenschappelijke laboratoriumapparatuur en vrij...

Video: Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Bioengineering

Live Cell Imaging during Mechanical Stretch

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in their capability for measuring responses in real time in live cells or viable tissue. A protocol was developed with the use of a cell actuator to distend live cells grown on or tissues attached to an elastic substrate while imaging with confocal and atomic force microscopy (AFM). Preliminary studies show that tonic stretching of human bronchial epithelial cells caused a significant increase in the production of mitochondrial superoxide. Moreover, using this protocol, alveolar epithelial cells were stretched and imaged, which showed direct damage to the epithelial cells by overdistention simulating one form of lung injury in vitro. A protocol to conduct AFM nano-indentation on stretched cells is also provided.

A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy.

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in ...

Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other physical or chemical stimuli. They have been commonly used for in vitro cell studies by providing in vivo like surroundings. Especially, how cells response to chemical gradients, electrical fields, and shear stresses has drawn many interests since these phenomena are important in understanding cellular properties and functions. These microfluidic chips can be made of glass substrates, silicon wafers, polydimethylsiloxane (PDMS) polymers, polymethylmethacrylate (PMMA) substrates, or polyethyleneterephthalate (PET) substrates. Out of these materials, PMMA substrates are cheap and can be easily processed using laser ablation and writing. Although a few microfluidic devices have been designed and fabricated for generating multiple, coexisting chemical and electrical stimuli, none of them was considered efficient enough in reducing experimental repeats, particular for screening purposes. In this report, we describe our design and fabrication of two PMMA-based microfluidic chips for investigating cellular responses, in the production of reactive oxygen species and the migration, under single or coexisting chemical/electrical/shear stress stimuli. The first chip generates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the culture regions, together with a shear stress gradient produced inside each of these areas. The second chip generates the same relative concentrations, but with five different electric field strengths created within each culture area. These devices not only provide cells with a precise, controllable micro-environment but also greatly increase the experimental throughput.

Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other ...

An Optimized O9-1/Hydrogel System for Studying Mechanical Signals in Neural Crest Cells

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to various organs and tissues. Tissue stiffness produces mechanical force, a physical cue that plays a critical role in NCC differentiation; however, the mechanism remains unclear. The method described here provides detailed information for the optimized generation of polyacrylamide hydrogels of varying stiffness, the accurate measurement of such stiffness, and the evaluation of the impact of mechanical signals in O9-1 cells, a NCC line that mimics in vivo NCCs.
Hydrogel stiffness was measured using atomic force microscopy (AFM) and indicated different stiffness levels accordingly. O9-1 NCCs cultured on hydrogels of varying stiffness showed different cell morphology and gene expression of stress fibers, which indicated varying biological effects caused by mechanical signal changes. Moreover, this established that varying the hydrogel stiffness resulted in an efficient in vitro system to manipulate mechanical signaling by altering gel stiffness and analyzing the molecular and genetic regulation in NCCs. O9-1 NCCs can differentiate into a wide range of cell types under the influence of the corresponding differentiation media, and it is convenient to manipulate chemical signals in vitro. Therefore, this in vitro system is a powerful tool to study the role of mechanical signaling in NCCs and its interaction with chemical signals, which will help researchers better understand the molecular and genetic mechanisms of neural crest development and diseases.

Detailed step-by-step protocols are described here for studying mechanical signals in vitro using multipotent O9-1 neural crest cells and polyacrylamide hydrogels of varying stiffness.

Neural crest cells (NCCs) are vertebrate embryonic multipotent cells that can migrate and differentiate into a wide array of cell types that give rise to ...

Developmental Biology

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the F&#246;rster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Here, we present a protocol for the simultaneous use of F&#246;rster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...

Pattern Generation for Micropattern Traction Microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both force-sensing molecules at adhesion sites, and force-dependent transcription factors that regulate lineage-specific gene expression and drive phenotypic outputs. However, the signaling networks converting mechanical tension into biochemical pathways have remained elusive. To explore the signaling pathways engaged upon mechanical tension applied to cell surface receptor, superparamagnetic microbeads can be used. Here we present a protocol for using magnetic beads to apply forces to cell surface adhesion proteins. Using this approach, it is possible to investigate not only force-dependent cytoplasmic signaling pathways by various biochemical approaches, but also adhesion remodeling by magnetic isolation of adhesion complexes attached to the ligand-coated beads. This protocol includes the preparation of ligand-coated superparamagnetic beads, and the application of define tensile forces followed by biochemical analyses. Additionally, we provide a representative sample of data demonstrating that tension applied to integrin-based adhesion triggers adhesion remodeling and alters protein tyrosine phosphorylation.

Cell surface adhesions are central in mechanotransduction, as they transmit mechanical tension and initiate the signaling pathways involved in tissue homeostasis and development. Here, we present a protocol for dissecting the biochemical pathways that are activated in response to tension, using ligand-coated magnetic microbeads and force application to adhesion receptors.

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both ...

Biology

Quantitative Analysis of Cell Edge Dynamics during Cell Spreading

Cell spreading is a dynamic process in which a cell suspended in media attaches to a substrate and flattens itself from a rounded to a thin and spread-out shape. Following the cell-substrate attachment, the cell forms a thin sheet of lamellipodia emanating from the cell body. In the lamellipodia, globular actin (G-actin) monomers polymerize into a dense filamentous actin (F-actin) meshwork that pushes against the plasma membrane, thereby providing the mechanical forces required for the cell to spread. Notably, the molecular players that control the actin polymerization in lamellipodia are essential for many other cellular processes, such as cell migration and endocytosis. 
Since spreading cells form continuous lamellipodia that span the entire cell periphery and persistently expand outward, cell spreading assays have become an efficient tool to assess the kinetics of lamellipodial protrusions. Although several technical implementations of the cell spreading assay have been developed, a detailed description of the workflow, which would include both a step-by-step protocol and computational tools for data analysis, is currently lacking. Here, we describe the experimental procedures of the cell spreading assay and present an open-source tool for quantitative and unbiased analysis of cell edge dynamics during spreading. When combined with pharmacological manipulations and/or gene-silencing techniques, this protocol is amenable to a large-scale screen of molecular players regulating lamellipodial protrusions.

In this protocol, we present the experimental procedures of a cell spreading assay that is based on live-cell microscopy. We provide an open-source computational tool for the unbiased segmentation of fluorescently labeled cells and quantitative analysis of lamellipodia dynamics during cell spreading.

Cell spreading is a dynamic process in which a cell suspended in media attaches to a substrate and flattens itself from a rounded to a thin and spread-out ...

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 ...

Cytoskeleton and Focal Adhesion Organization Assay: An Immunofluorescence-based Method to Study Cell Adhesion and Spreading on Substrates

Source: Ali, M. Y et al., <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/52532"> Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry.</a> J. Vis. Exp. (2015)
This video describes the technique of studying the cytoskeleton and focal adhesions in primary human colon cancer cells to understand its variations depending on substrate rigidity. This culture of cancer cells on soft and hard substrates allows various downstream biophysical measurements for cancer prognosis.

Source: Ali, M. Y et al.,  Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for ...

Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress

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