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-st...

<ol>
	<li>Geiger, B., Bershadsky, A., Pankov, R., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmembrane+crosstalk+between+the+extracellular+matrix--cytoskeleton+crosstalk.">Transmembrane crosstalk between the extracellular matrix--cytoskeleton crosstalk.</a> Nature Reviews: Molecular Cell Biology. 2 (11), 793-805 (2001).</li><li>Geiger, B., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+architecture+and+function+of+matrix+adhesions.">Molecular architecture and function of matrix adhesions.</a> Cold Spring Harbor Perspectives in Biology. 3 (5), (2011).</li><li>Lawson, C. D., Burridge, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+on-off+relationship+of+Rho+and+Rac+during+integrin-mediated+adhesion+and+cell+migration.">The on-off relationship of Rho and Rac during integrin-mediated adhesion and cell migration.</a> Small GTPases. 5, e27958 (2014).</li><li>Heo, J., Campbell, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+redox-mediated+guanine+nucleotide+exchange+on+redox-active+Rho+GTPases.">Mechanism of redox-mediated guanine nucleotide exchange on redox-active Rho GTPases.</a> Journal of Biological Chemistry. 280 (35), 31003-31010 (2005).</li><li>Tolbert, C. E., Beck, M. V., Kilmer, C. E., Srougi, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+ATM+positively+regulates+Rac1+activity+and+cellular+migration+through+oxidative+stress.">Loss of ATM positively regulates Rac1 activity and cellular migration through oxidative stress.</a> Biochemical and Biophysical Research Communications. 508 (4), 1155-1161 (2019).</li><li>Hobbs, G. 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=Redox+regulation+of+Rac1+by+thiol+oxidation.">Redox regulation of Rac1 by thiol oxidation.</a> Free Radical Biology and Medicine. 79, 237-250 (2015).</li><li>Zhang, Y., 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=Mitochondrial+redox+sensing+by+the+kinase+ATM+maintains+cellular+antioxidant+capacity.">Mitochondrial redox sensing by the kinase ATM maintains cellular antioxidant capacity.</a> Science Signaling. 11 (538), (2018).</li><li>Hobbs, G. A., Zhou, B., Cox, A. D., Campbell, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho+GTPases,+oxidation,+and+cell+redox+control.">Rho GTPases, oxidation, and cell redox control.</a> Small GTPases. 5, e28579 (2014).</li><li>Shiloh, Y., Ziv, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ATM+protein+kinase:+regulating+the+cellular+response+to+genotoxic+stress,+and+more.">The ATM protein kinase: regulating the cellular response to genotoxic stress, and more.</a> Nature Reviews: Molecular Cell Biology. 14 (4), 197-210 (2013).</li><li>Lang, L., 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=ATM-Mediated+Phosphorylation+of+Cortactin+Involved+in+Actin+Polymerization+Promotes+Breast+Cancer+Cells+Migration+and+Invasion.">ATM-Mediated Phosphorylation of Cortactin Involved in Actin Polymerization Promotes Breast Cancer Cells Migration and Invasion.</a> Cellular Physiology and Biochemistry. 51 (6), 2972-2988 (2018).</li><li>Peter, Y., 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=Elevated+Cu/Zn-SOD+exacerbates+radiation+sensitivity+and+hematopoietic+abnormalities+of+Atm-deficient+mice.">Elevated Cu/Zn-SOD exacerbates radiation sensitivity and hematopoietic abnormalities of Atm-deficient mice.</a> European Molecular Biology Organization Journal. 20 (7), 1538-1546 (2001).</li><li>Takao, N., Li, Y., Yamamoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+roles+for+ATM+in+cellular+response+to+oxidative+stress.">Protective roles for ATM in cellular response to oxidative stress.</a> Federation of European Biochemical Societies Letters. 472 (1), 133-136 (2000).</li><li>Jansen, S., Gosens, R., Wieland, T., Schmidt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paving+the+Rho+in+cancer+metastasis:+Rho+GTPases+and+beyond.">Paving the Rho in cancer metastasis: Rho GTPases and beyond.</a> Pharmacology &amp; Therapeutics. 183, 1-21 (2018).</li><li>Berrier, A. L., Martinez, R., Bokoch, G. M., LaFlamme, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+integrin+beta+tail+is+required+and+sufficient+to+regulate+adhesion+signaling+to+Rac1.">The integrin beta tail is required and sufficient to regulate adhesion signaling to Rac1.</a> Journal of Cell Science. 115 (Pt 22), 4285-4291 (2002).</li><li>Arthur, W. T., Petch, L. A., Burridge, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+engagement+suppresses+RhoA+activity+via+a+c-Src-dependent+mechanism.">Integrin engagement suppresses RhoA activity via a c-Src-dependent mechanism.</a> Current Biology. 10 (12), 719-722 (2000).</li><li>Arthur, W. T., Burridge, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RhoA+inactivation+by+p190RhoGAP+regulates+cell+spreading+and+migration+by+promoting+membrane+protrusion+and+polarity.">RhoA inactivation by p190RhoGAP regulates cell spreading and migration by promoting membrane protrusion and polarity.</a> Molecular Biology of the Cell. 12 (9), 2711-2720 (2001).</li><li>Chandra, S., Kalaivani, R., Kumar, M., Srinivasan, N., Sarkar, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sendai+virus+recruits+cellular+villin+to+remodel+actin+cytoskeleton+during+fusion+with+hepatocytes.">Sendai virus recruits cellular villin to remodel actin cytoskeleton during fusion with hepatocytes.</a> Molecular Biology of the Cell. 28 (26), 3801-3814 (2017).</li><li>Fitzpatrick, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Measuring Cell Fluorescence Using ImageJ. , (2014).</li><li>Berginski, M. E., Vitriol, E. A., Hahn, K. M., Gomez, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+quantification+of+focal+adhesion+spatiotemporal+dynamics+in+living+cells.">High-resolution quantification of focal adhesion spatiotemporal dynamics in living cells.</a> PLoS One. 6 (7), e22025 (2011).</li><li>Horzum, U., Ozdil, B., Pesen-Okvur, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Step-by-step+quantitative+analysis+of+focal+adhesions.">Step-by-step quantitative analysis of focal adhesions.</a> MethodsX. 1, 56-59 (2014).</li><li>Elosegui-Artola, 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=Image+analysis+for+the+quantitative+comparison+of+stress+fibers+and+focal+adhesions.">Image analysis for the quantitative comparison of stress fibers and focal adhesions.</a> PLoS One. 9 (9), e107393 (2014).</li><li>Meller, J., Vidali, L., Schwartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+RhoG+is+dispensable+for+integrin-mediated+cell+spreading+but+contributes+to+Rac-independent+migration.">Endogenous RhoG is dispensable for integrin-mediated cell spreading but contributes to Rac-independent migration.</a> Journal of Cell Science. 121 (Pt 12), 1981-1989 (2008).</li><li>Donaldson, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+Staining.">Immunofluorescence Staining.</a> Current Protocols in Cell Biology. 69 (43), 1-7 (2015).</li><li>Burry, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controls+for+immunocytochemistry:+an+update.">Controls for immunocytochemistry: an update.</a> Journal of Histochemistry and Cytochemistry. 59 (1), 6-12 (2011).</li><li>Grashoff, C., 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=Measuring+mechanical+tension+across+vinculin+reveals+regulation+of+focal+adhesion+dynamics.">Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics.</a> Nature. 466 (7303), 263-266 (2010).</li><li>Kumar, 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=Correction:+Talin+tension+sensor+reveals+novel+features+of+focal+adhesion+force+transmission+and+mechanosensitivity.">Correction: Talin tension sensor reveals novel features of focal adhesion force transmission and mechanosensitivity.</a> Journal of Cell Biology. 214 (2), 231 (2016).</li><li>Kumar, 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=Talin+tension+sensor+reveals+novel+features+of+focal+adhesion+force+transmission+and+mechanosensitivity.">Talin tension sensor reveals novel features of focal adhesion force transmission and mechanosensitivity.</a> Journal of Cell Biology. 213 (3), 371-383 (2016).</li><li>Friedrichs, J., Helenius, J., Muller, D. 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 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 at...

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&#176;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&#39;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 &#181;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>
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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 cell...

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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 ...

This method will enable researchers to quantify the early dynamics of cellular adhesion and spreading of anchorage-dependent cells onto the extracellular matrix protein fibronectin during oxidative stress. This technique is versatile and can be adapted to examine cytoskeletal dynamics under a wide range of conditions. Furthermore, data acquisition and analysis are performed using common laboratory equipment and freely available software.Understanding the mechanisms for how cells attach and spread on the ECM during alterations in redox status can provide valuable insight into normal and disease states, such as metastatic cancer. This method can be used to examine cell spreading and adhesion under different cell culture conditions, anchorage-dependent cell lines, and/oxidants to answer a broad range of biological questions. There are numerous reagents required to perform this technique.Therefore, it is essential that all solutions are made in advance before beginning. In a BSL-II certified laminar flow hood set out a tissue culture-certified 24-well plate. Place one glass coverslip into each well.Label the plate according to figure 1B of the text protocol. Next, pipette 500 microliters of the fibronectin solution into each well. Pipette the solution over each coverslip a few times to ensure an even coating and complete submersion.Cover the plate with the lid. Incubate the plate at 37 degrees Celsius with 5%carbon dioxide for one hour. Then remove the plate from the incubator and aspirate the fibronectin solution from the wells.Wash the wells three times using 500 microliters of 1X PBS per wash. Aspirate the final wash of 1X PBS, and block wells with 500 microliters of a 0.5%delipidated BSA solution at 37 degrees Celsius with 5%carbon dioxide for a minimum of 15 minutes. First, pre-warm the needed solutions in a water bath at 37 degrees Celsius.In a BSL-II certified laminar flow hood, start with a confluent monolayer of REF52 cells in a 10-square centimeter dish and wash the cells twice with six milliliters of warm 1X PBS. Serum starve the cells for at least one hour in six milliliters of warm delipidated BSA solution. Next, wash the cells with six milliliters of warmed 1X PBS.Aspirate the PBS and add 1.5 milliliters of warm 0.5%trypsin-EDTA solution. Incubate the cells at 37 degrees Celsius with 5%carbon dioxide for approximately two minutes. Observe the cells under a light microscope to ensure that the detachment is complete.If the cells are still adherent after tapping the plate on the bench top, return them to the incubator for an additional two minutes. Pipette 1.5 milliliters 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 of the remaining adherent cells.If the cells appear clumpy, further triturate the cell suspension by gently pipetting up and down over the back of the dish. Next, count the cells using trypan blue exclusion in an automated cell counter. Remove an appropriate amount of cells to create a cell suspension with the cell density between 10, 000 and 30, 000 cells per milliliter in delipidated BSA in a 15-milliliter conical tube.Use a fixed-angle rotor in a tabletop centrifuge to centrifuge the cells at 300 times g for five minutes. Aspirate the supernatant and re-suspend the cell pellet in seven milliliters of warm delipidated BSA solution. Then evenly divide the cell suspension into two 15-milliliter conical tubes, one for vehicle-alone control, and one for Ku55933.Using a tube rotator, revolve the tubes for 90 to 120 minutes in a cell culture incubator at 37 degrees Celsius. 30 minutes before plating, add Ku55933 and DMSO to each tube to a final concentration of 10 micromolar. Return the cell suspension to the rotator for the remaining time.Immediately prior to plating the cells, retrieve the plate from the incubator and aspirate the delipidated BSA solution. After this, remove 500 microliters of the cell suspension from each treatment group and add each one to one of the fibronectin-coated cover slips in the 24-well plate. Continue incubating the plate and the cell suspension at the previous conditions.Then allow the cells to adhere to the coverslip for the desired length of time. After the desired time for adhesion has passed, aspirate the cell solution from each coverslip in the plate. Gently dispense 500 microliters of 3.7%paraformaldehyde solution onto each coverslip by pipetting down the sides of the wells, and wait 10 to 15 minutes.Next, remove the paraformaldehyde solution and wash each coverslip twice with 1X PBS using 500 microliters of PBS per wash. Aspirate the PBS and permeabilized cells on each coverslip with 500 microliters of 0.2%Triton X-100 and 1X PBS for 10 to 15 minutes at room temperature. Then wash each coverslip three times with 1X PBS using 500 microliters of PBS per wash.Block the cells on each coverslip with 500 microliters of immunofluorescence blocking buffer for 30 to 60 minutes. Dilute the primary anti-paxillin antibody in the blocking buffer at a ratio of one-to-250. Mix well and add 200 microliters of the antibody solution to each coverslip.Incubate at room temperature for at least one hour. After this, aspirate the antibody solution and wash each coverslip with 500 microliters of 1X PBS for 10 minutes. Protect the samples from light from this point forward.Dilute the phalloidin F-actin probe conjugated to the red fluorescent Alexa 594 dye and the goat anti-mouse 488 fluorescent secondary antibody in the same blocking buffer solution. Mix well and add 200 microliters of the antibody solution to each coverslip for 30 minutes. Aspirate the antibody solution and wash each coverslip with 500 microliters of 1X PBS for 10 minutes.Aspirate the PBS and rinse the cover slips once with 500 microliters of deionized water. Then fix the cover slips onto microscope slides using anti-fade mounting medium containing DAPI. After fixation and staining with an anti-paxillin antibody, fluorescent 8-bit grayscale images of the REF52 cells are acquired.Upon completion of all image processing steps, individual focal adhesions should be prominent, in focus, and readily distinguishable from one another. Grayscale fluorescence images of anti-paxillin and phalloidin F-actin probe stained cells are then taken after being plated on fibronectin. Prominent focal adhesions and stress fibers should be readily visible in REF52 cells after being allowed to adhere on fibronectin for 20 to 30 minutes.F-actin enriched ruffles at the leading edge of cell membranes are indicated with an arrow. Similar fluorescent images of phalloidin F-actin probe and anti-paxillin staining are analyzed for the percentage of stress fibers in cell spreading. Notably, oxidant treatment causes a significant increase in stress fiber formation at all adhesion time points examined, and a decrease in cell spreading following 15 minutes of cell adhesion to fibronectin.Plating at higher cell densities leads to cellular crowding which prohibits cells from fully spreading due to overconfluency. Notice that the cell edges are indistinguishable from adjacent cells. As a result, quantification of individual cells is precluded and spreading circumference cannot be accurately determined.A separate cell line of mouse embryonic fibroblasts are held in suspension and then plated on fibronectin for 30 minutes. Cells were then fixed and stained with an anti-paxillin antibody. Note the out-of-focus cells.Furthermore, the cross-reactivity of the anti-paxillin antibody with cellular debris will alter thresholding during quantitative image analysis and should not be included in the analysis. During image processing, use the look-up tool in Fiji to examine the image histogram while adjusting the brightness/contrast to avoid pitfalls related to signal saturation. Changes in adhesion and spreading may influence cell migration.Methods to track single cell migration, wound healing, or chemotaxis invasion assays can determine if alterations in migration are occurring. Rho family GTPases regulate cell adhesion dynamics through their specific spatial temporal activation. Phenotypic changes in adhesion and spreading during oxidative stress may be suggestive of downstream regulatory roles for these proteins.This method requires the use of BSL-II cell lines and hazardous chemical reagents. Researchers require chemical and biological safety training as determined by their institution's environmental health and safety office.

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Research

JoVE Journal

Biochemistry

7.3K visualizações.  University of North Carolina at Chapel Hill. Este método permitirá aos pesquisadores quantificar a dinâmica inicial da adesão celular e a disseminação de células dependentes de ancoragem na fibronectina de proteínas da matriz extracelular durante o estresse oxidativo. Esta técnica é versátil e pode ser adaptada para examinar a dinâmica citoesquelletal sob uma ampla gama de condições. Além disso, a aquisição e análise de dados são realizadas utilizando equipamentos de laboratório comuns e softwares disponíveis livrem...