1. Immunofluorescence Microscopy Assays
<ol>
	<li>Take the substrates with cells to be immunostained to the laminar hood and remove the culture media.</li>
	<li>Rinse the substrates with PBS and fix the cells with 4% paraformaldehyde in PBS for 20 min at RT.</li>
	<li>Wash the substrates with PBS three times (5 min each time). Consequently, incubate the substrates with 500 &#181;l signal enhancer for 30 min and rinse with PBS.</li>
	<li>Incubate cells with the monoclonal anti-vinculin antibody at a 1:250 dilution in PBS for 45 min at RT. Then, wash the substrates with PBS 3 times (5 min each time).</li>
	<li>Incubate the samples with secondary antibody Alexa Fluor 488 goat anti-mouse IgG at a 1:200 dilution in PBS at RT for 30 min. Wash the substrates with PBS 3 times (5 min each time).</li>
	<li>To visualize the F-actin structure, incubate cells with TRITC phalloidin conjugates at a concentration of 50 &#181;g/ml for 45 min at RT. Then, wash the substrates with PBS 3 times (5 min each time).</li>
	<li>Image the samples using a confocal scanning laser microscope (Figure 1A-1D).</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>&#160;Lonza</td>
			<td>&#160;17-516F</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde&#160;&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>RT15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Signal enhancer</td>
			<td>Life technologies</td>
			<td>&#160; I36933</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal anti vinculin antibody&#160;&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>V9131</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-mouse IgG&#160;&#160;</td>
			<td>Life technologies</td>
			<td>A11001</td>
			<td></td>
		</tr>
		<tr>
			<td>TRITC phalloidin conjugates&#160;&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P1951</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>12 mm2 glass cover slips&#160;&#160;</td>
			<td>Corning</td>
			<td>2865-12</td>
			<td></td>
		</tr>
	</tbody>
</table>

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/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.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/20345/20345fig1.jpg" /> 
Figure 1. Immunofluorescent staining of F-actin and vinculin on soft gel and hard polystyrene substrates.&#160;No actin fiber was present in the less spread (A1) tumor cell or (A2) normal cell on soft 2 kPa gels. However, punctate vinculin-containing focal adhesions are present on soft gels (B1-B2). Conversely, primar...

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

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 ...

- Substrate morphology greatly influences cancer cells&#39; behavior and metastatic responses. A dynamic network of actin cytoskeleton provides structural support to cells and mediates cellular movement. Large membrane-associated protein complexes called focal adhesions connect the cytoskeleton with the extracellular matrix, facilitating cell-spreading.
To begin staining, take a cancer cell culture, and treat them with paraformaldehyde to fix and permeabilize the cells. Now, incubate the cells with a signal enhancer reagent containing proteins to prepare the cells to reduce background staining. Add anti-vinculin primary antibodies that bind to vinculin, one of the proteins within the focal adhesion complex.
Next, incubate the sample with a fluorescent dye conjugated secondary antibody targeting the primary antibody. Finally, treat the cells with fluorescent tagged phalloidin conjugates that selectively bind to actin filaments of the cytoskeleton. Observe the cells under a confocal laser scanning microscope to study their fluorescent signals.
Cells with well-spread morphology exhibit defined actin fibers and present distinct and elongated vinculin-containing focal adhesions. Conversely, poorly spread cells do not possess defined actin filaments, but display punctate vinculin-containing focal adhesions. In the following protocol, we will demonstrate an immunofluorescence assay to study cytoskeleton and focal adhesion organization in primary colon cancer cells.
- Take the substrates to be immunostained to the laminar hood and remove the culture media. Then, rinse the substrates with phosphate-buffered saline, or PBS, and fix the cells with 4% paraformaldehyde in PBS for 20 minutes at room temperature. Wash the substrates with PBS for five minutes a total of three times. Then, incubate the substrates with 500 microliters of signal enhancer for 30 minutes before rinsing with PBS.
Next, incubate the cells with monoclonal anti-vinculin antibody at a 1 to 250 dilution in PBS for 45 minutes at room temperature. Then, wash the substrates with PBS for five minutes a total of three times. Incubate the samples with a secondary antibody Alexa Fluor 488 goat anti-mouse IgG at a 1 to 200 dilution in PBS at room temperature for 30 minutes. Then, wash the substrates again with PBS for five minutes a total of three times.
To visualize the F-actin structure, incubate the cells with tetramethylrhodamine-phalloidin conjugates at a concentration of 50 microliters per milliliter for 45 minutes at room temperature. Then, wash the substrates again as before. Finally, proceed to image the samples using a confocal scanning laser microscope.

cytoskeleton-focal-adhesion-organization-assay-an-immunofluorescence

Research

JoVE Encyclopedia of Experiments

Cancer Research

Colorectal Cancer

Assays & techniques

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.

Overview

Protocol

<img title="figure-results-58" alt="figure-results-58" class="xfigimg" src="/files/ftp_upload/20345/20345fig1.jpg" /> 
Figure 1. Immunofluorescent staining of F-actin and vinculin on soft gel and hard polystyrene substrates.&#160;No actin fiber was present in the less spread (A1) tumor cell or (A2) normal cell on soft 2 kPa gels. However, punctate vinculin-containing focal adhesions are present on soft gels (B1-B2). Conversely, primar...

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

In This Article

Overview

Protocol

Results

Disclosures

Materials

Name	Company	Catalog Number	Comments
Reagents
PBS	Lonza	17-516F
Paraformaldehyde	Electron Microscopy Sciences	RT15710
Signal enhancer	Life technologies	I36933
Monoclonal anti vinculin antibody	Sigma-Aldrich	V9131
Alexa Fluor 488 goat anti-mouse IgG	Life technologies	A11001
TRITC phalloidin conjugates	Sigma-Aldrich	P1951
Materials
12 mm2 glass cover slips	Corning	2865-12