This work was supported by the Polish National Science Center under grant no. 2014/14/M/NZ1/00437.

1. Preparatory steps
<ol>
	<li>Cell seeding to obtain confluent monolayers
	<ol>
		<li>Before seeding, coat the wells of a 4-wells chamber slide with collagen I (or other ECM component of choice). For collagen I coating, follow a commercial protocol: https://www.sigmaaldrich.com/technical-documents/articles/biofiles/collagen-product-protocols.html at a concentration of 8 &#956;g/cm2.</li>
		<li>Seed 400,000 cells to one well of a 4-well chamber slide.</li>
		<li>Culture the cells for 24 h (or longer, dependi...

<ol>
	<li>Li, D. S., Zimmermann, J., Levine, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+closure+of+circular+wounds+through+coordinated+collective+motion.">Modeling closure of circular wounds through coordinated collective motion.</a> Search Results. 13 (1), 016006 (2016).</li><li>Ilina, O., Friedl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+collective+cell+migration+at+a+glance.">Mechanisms of collective cell migration at a glance.</a> Journal of Cell Science. 122, 3203-3208 (2009).</li><li>Ladoux, B., Mege, R. 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B. C., Kurniawan, N. A., Bouten, C. V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+quantitative+analysis+of+cell,+nucleus+and+focal+adhesion+morphology.">An automated quantitative analysis of cell, nucleus and focal adhesion morphology.</a> PLoS One. 13 (3), 0195201 (2018).</li><li>Fokkelman, M., 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=Cellular+adhesome+screen+identifies+critical+modulators+of+focal+adhesion+dynamics,+cellular+traction+forces+and+cell+migration+behaviour.">Cellular adhesome screen identifies critical modulators of focal adhesion dynamics, cellular traction forces and cell migration behaviour.</a> Scientific Reports. 6, 31707 (2016).</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>Kim, D. H., Wirtz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+adhesion+size+uniquely+predicts+cell+migration.">Focal adhesion size uniquely predicts cell migration.</a> FASEB Journal. 27 (4), 1351-1361 (2013).</li><li>Pincus, Z., Theriot, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+quantitative+methods+for+cell-shape+analysis.">Comparison of quantitative methods for cell-shape analysis.</a> Journal of Microscopy. 227, 140-156 (2007).</li><li>Tsygankov, D., 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=CellGeo:+a+computational+platform+for+the+analysis+of+shape+changes+in+cells+with+complex+geometries.">CellGeo: a computational platform for the analysis of shape changes in cells with complex geometries.</a> Journal of Cell Biology. 204 (3), 443-460 (2014).</li><li>Tiryaki, V. M., Adia-Nimuwa, U., Ayres, V. M., Ahmed, I., Shreiber, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Texture-based+segmentation+and+a+new+cell+shape+index+for+quantitative+analysis+of+cell+spreading+in+AFM+images.">Texture-based segmentation and a new cell shape index for quantitative analysis of cell spreading in AFM images.</a> Cytometry A. 87 (12), 1090-1100 (2015).</li><li>Tong, J., 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=Cell+micropatterning+reveals+the+modulatory+effect+of+cell+shape+on+proliferation+through+intracellular+calcium+transients.">Cell micropatterning reveals the modulatory effect of cell shape on proliferation through intracellular calcium transients.</a> Biochimica et Biophysica Acta. 1864 (12), 2389-2401 (2017).</li><li>Vartanian, K. B., Kirkpatrick, S. J., Hanson, S. R., Hinds, M. 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The authors have nothing to disclose.

Cell-cell and cell-substrate adhesion constitute inherent attributes of the epithelial cells and play the critical role in tissue morphogenesis and embriogenesis. In adult tissues the proper regulation of mechanical properties of the cell layer is crucial in maintaining homeostasis and preventing pathological responses like tumor progression and metastasis. The size and number of focal adhesions depend on the strength of cell-substrate adhesion, while cell shape depends on contractile forces and is related to the status ...

Epithelial cell monolayers act as a collective in which cell-cell and cell-substrate adhesion as well as contractile forces and tensions represent important parameters and their proper balance contributes to the overall integrity of the unit1,2,3. Thus, assessing these parameters represents a way to establish the current status of the cell layer.
The two methods described here represent a two-dimensional analysis of the confluent monolayers of adherent, epithelial cells (in this case MCF7 breast cancer cell line). The analysis is performed using confocal images (single Z-slices) from different regions on the Z-axis; basal region near a substrate for focal adhesion (FA) measurements and apical region for cell shape measurements. The presented methods are relatively simple and require standard laboratory techniques and open-source software. Confocal microscopy is sufficient for this protocol, so it can be performed without employing more specialized TIRF (Total Internal Reflection Fluorescence) microscopy. Thus, the protocol could be implemented in a relatively standard laboratory setting. Although the accuracy of the methods is limited, they can distinguish basic differences in focal adhesion and cell shape.
Both methods described here consist of the standard experimental procedures such as cell culturing, immunostaining, confocal imaging and image analysis performed using ImageJ. However, any image processing software with the appropriate functions can be used. The presented methods can track and compare changes inflicted by pharmacological treatment or minimal genetic modification. Obtaining definite values is not recommended, due to the limited precision of these methods. Two automated macros were included, to facilitate the measurements of many images.

<table>
	<tbody>
		<tr>
			<td>Alexa Fluor 594</td>
			<td>ThermoFisher Scientific</td>
			<td>A32740</td>
			<td>goat anti-rabbit, 1:500</td>
		</tr>
		<tr>
			<td>Ammonium chloride</td>
			<td>Sigma</td>
			<td>A9434</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>BioShop</td>
			<td>ALB001.500</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen from calf skin</td>
			<td>Sigma</td>
			<td>C9791-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td>1:10000 (stock 1 mg/mL in H2O), nucleic acid staining</td>
		</tr>
		<tr>
			<td>DMEM + GlutaMAX, 1 g/L D-Glucose, Pyruvate</td>
			<td>ThermoFisher Scientific</td>
			<td>21885-025</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>ThermoFisher Scientific</td>
			<td>10270-136</td>
			<td></td>
		</tr>
		<tr>
			<td>Junction plakoglobin</td>
			<td>Cell Signaling</td>
			<td>2309S</td>
			<td>rabbit, 1:400</td>
		</tr>
		<tr>
			<td>Laminar-flow cabinet class 2</td>
			<td>Alpina</td>
			<td></td>
			<td>standard equipment</td>
		</tr>
		<tr>
			<td>MCF7-basedHAX1KD cell line</td>
			<td>Cell line established in the National Institute of Oncology, Warsaw, described in Balcerak et al., 2019</td>
			<td></td>
			<td>MCF7 cell line withHAX1knockdown</td>
		</tr>
		<tr>
			<td>MCF7 cell line (CONTROL)</td>
			<td>ATCC</td>
			<td>ATCC HTB-22</td>
			<td>epithelial, adherent breast cancer cell line</td>
		</tr>
		<tr>
			<td>Olympus CK2 light microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paxillin</td>
			<td>Abcam</td>
			<td>ab32084</td>
			<td>rabbit, 1:250, Y113</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>ThermoFisher Scientific</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin-TRITC conjugate</td>
			<td>Sigma</td>
			<td>P1951</td>
			<td>1:400 (stock 5 mg/mL in DMSO), actin labeling</td>
		</tr>
		<tr>
			<td>PTX</td>
			<td>Sigma</td>
			<td>T7402-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>TBST &#8211; NaCl</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>TBST &#8211; Trizma base</td>
			<td>Sigma</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>9002-93-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss LSM800 Confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of cell-substrate adhesion area and cell shape distributions in mcf7 cell monolayers

The methods presented here quantify some parameters of confluent adherent cell monolayers from multiple appropriately stained confocal images: adhesion to the substrate as a function of the number and size of focal adhesions, and cell shape, characterized by the cell shape index and other shape descriptors. Focal adhesions were visualized by paxillin staining and cell-cell borders were marked by junction plakoglobin and actin. The methods for cell culture and staining were standard; images represent single focal planes; image analysis was performed using publicly available image processing software. The presented protocols are used to quantify the number and size of focal adhesions and the differences in cell shape distribution in the monolayers, but they can be repurposed for the quantification of the size and shape of any other distinct cellular structure that can be stained (e.g., mitochondria or nuclei). Assessing these parameters is important in the characterization of the dynamic forces in adherent cell layer, including cell adhesion and actomyosin contractility that affects cell shape.

Focal adhesion analysis 
The knockdown of HAX1 gene was previously shown to affect focal adhesions6. Cells were cultured on collagen I-coated surface for 48 h. Images of the MCF7 control cells and MCF7 cells with a HAX1 knockdown (HAX1 KD) from three independent experiments stained with focal adhesion protein paxillin were obtained using a confocal microscope (image from single focal plane/Z-slice from basal region). FAs from about 2,000-2,500 cells...

Watch this Scientific Journal Video about Quantification of Cell-Substrate Adhesion Area and Cell Shape Distributions in MCF7 Cell Monolayers at JoVE.com

Quantification of Cell-Substrate Adhesion Area and Cell Shape Distributions in MCF7 Cell Monolayers

The article describes quantification of 1) the size and number of focal adhesions and 2) cell shape index and its distribution from confocal images of the confluent monolayers of MCF7 cells.

The methods presented here quantify some parameters of confluent adherent cell monolayers from multiple appropriately stained confocal images: adhesion to ...

This protocol enables quantification of the area, perimeter, and shape of intracellular structure from its two-dimensional image. This technique is quick and easy, requires only standard laboratory settings and a confocal microscope and uses opensource software. Demonstrating the procedure will be Anna Balcerak, a PhD student from my laboratory.Begin by seeding four times 10 to the fifth cells in 0.5 milliliters of culture medium per well in a four-well collagen one coated chamber slide for a 24-hour culture in a 37 degree Celsius and five degree carbon dioxide incubator. The next morning, use an optical inverted microscope to verify the presence of at least 90%confluent monolayer and use a confocal microscope to obtain single Z-slice images. For focal adhesion analysis, open the images in ImageJ and select analyze, set scale, remove scale, and global to set the image scale in pixels.To include the filename and the area of the region of interest in the measurement options, click analyze and set measurements and check the area and display label options. To subtract the background, select process and subtract background. Set the rolling ball radius to 50 pixels and check sliding paraboloid.To determine the area of the smallest region of interest, outline the smallest single focal adhesion and click analyze and measure to measure the area. When at least 20 regions of interest have been selected and measured, calculate and save the mean of the obtained results. Set the upper boundary to 25%of a typical cell area.To confer the image to binary, click image, adjust, and threshold, and check default, black and white, and dark background. To measure the number and areas of the regions of interest, select analyze and analyze particle and check pixel units, display results, clear results, and summarize. Transfer the slice, count, and total area average size data from the summary window to the data managing program of choice.For focal adhesion quantification, open the focal adhesion ImageJ macro and enter the area of the smallest region of interest as the area of the smallest region of interest parameter. Set the threshold type value to manual or auto and save the changes. Then call the macro from ImageJ and select the image to be processed.For manual cell shape analysis, open an image in an appropriate image processing software program and select the parameters to be measured. Use the freehand selection tool to manually delineate the cell borders as marked by the junction proteins of choice. The chosen parameters will be automatically calculated for each cell.When all of the cells have been outlined, select edit, selection, and add to manager. Only complete entirely visible cells with uninterrupted borders should be selected. To make the measurement, mark all of the numbers appearing in the left box of the region of interest manager and click measure.The results will appear in the results box and can be imported to the spreadsheet of choice. Automated cell analysis facilitates quantification of the large number of cells. For every new cell type, first run the macro to establish parameters.When the macro has finished, select set cell size boundaries. Click the label of the smallest and largest cells and click measure. Set the value of the smallest cell and the biggest cell variables and save the changes.Close all of the macro windows and select the image to be processed in grayscale. Then run the macro again. The macro will provide a table of the results that includes the cell shape index, aspect ratio, and regions of interest selections list data.Here, representative images of focal adhesions counted with ImageJ including the final numbered outlines and the overlays of the focal adhesion outlines with the original image for both the control and knockdown cell lines can be observed. As illustrated in this analysis, the knockdown cells demonstrated a higher number of adhesions per cell as well as adhesions that are larger in size compared to control cell line cells. In these images, representative regions of untreated and chemotherapeutically treated cell monolayers are shown.The outline cells were characterized according to their cell shape index values, for example in this analysis showing an increase in the last bin and a flattening of the main peaks for the drug-treated cells compared to untreated controls. A frequency distribution and cumulative distribution could then be generated for each cell culture. Grayscale imaging of the monolayers as demonstrated allowed the analysis and quantification of 512 cells from 12 fields of view revealing a relative frequency distribution of the cell shape index.For this protocol to work, it is important to use images of the best possible quality. Proper cell seeding, staining, and imaging should ensure images of a sufficient experimental quality.

quantification-cell-substrate-adhesion-area-cell-shape-distributions

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Biology

8.1K Views.  Maria Sklodowska-Curie National Research Institute of Oncology. This protocol enables quantification of the area, perimeter, and shape of intracellular structure from its two-dimensional image. This technique is quick and easy, requires only standard laboratory settings and a confocal microscope and uses opensource software. Demonstrating the procedure will be Anna Balcerak, a PhD student from my laboratory.Begin by seeding four times 10 to the fifth cells in 0.5 milliliters of culture medium per well in a four-well collagen one coated chamber slid...