Name: quantitation of endothelial cell adhesiveness in vitro
Uploaded: 2015-06-18T15:00:00
Duration: 10 min 24 s
Description: Watch this Scientific Journal Video about Quantitation of Endothelial Cell Adhesiveness In Vitro at JoVE.com

We are very grateful to Simon Bouffler for his full support and to Public Health England for infrastructure support. This work was supported by Public Health England throughthe National Institute for Health Research (NIHR) grant. This report is work commissioned by the NIHR. The views expressed in this publication are those of the authors and not necessarily those of the NHS, NIHR or the Department of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

1. Preparation of Glass Coverslips
<ol>
	<li>Sterilize 12 mm diameter round glass coverslips by soaking them in 70% ethanol for at least 10 min with occasional agitation to ensure total exposure of all coverslips to the ethanol. Pour glass coverslips and ethanol into a sterile cell culture dish (either 6 cm or 10 cm diameter).</li>
	<li>With a pair of sterile fine 5B forceps, pick up and place each individual glass coverslip into a well of a 24 well cluster plate. Take care to ensure that the coverslips are not touching the sides of the well and are approximately in the middle of the well.</li>
	<li>Leave the un-covered 24 well cluster plate with glass coverslips to dry in the flow cabinet. This takes about 10 min</li>
	<li>During this period, prepare the coating mix by diluting 10 mg/ml human fibronectin in Hank&#8217;s Balanced Salt solution (HBSS) to 0.1 mg/ml.</li>
	<li>When the coverslips are dry, pipette 100 &#181;l of coating solution onto each glass coverslip so the solution covers only the glass without pooling around the outside of the well. Place the covered 24 well cluster plate in a cell culture incubator for at least an hour.</li>
	<li>Just before use, remove the coating solution. The same coating solution can be transferred to a sterile tube and used up to three times with no visible loss of efficacy.</li>
</ol>
2. Preparation of Endothelial Cell Monolayer
<ol>
	<li>Culture human coronary artery endothelial cells (EC) in medium at 37 &#176;C and 5% carbon dioxide.</li>
	<li>Aspirate off culture media and rinse EC monolayer with 10 ml HBSS</li>
	<li>Aspirate off HBSS and add 2 ml 0.5% Trypsin, 0.2%EDTA solution. Incubate at RT for approximately 5 min.</li>
	<li>When EC can be dislodged by a tap to the side of the flask, add 5 ml of Soybean trypsin inhibitor and with pipette squirt the surface of the flask to further dislodge the cells.</li>
	<li>Transfer cells into a 15 ml tube and centrifuge the tube at 200 x g for 5 min.</li>
	<li>Aspirate off the supernatant and resuspend the EC pellet in 5 ml of media. Count the cells with a haemocytometer and adjust the cell concentration to 50,000 EC per ml of EC media.</li>
	<li>Dispense 1 ml of the cell suspension into each well of the 24 well plate containing coated glass coverslips. Incubate cells O/N in cell culture incubator at 37 &#176;C plus 5% carbon dioxide.</li>
	<li>The cells are ready for use in experiments when a confluent monolayer is formed, within 3 days.</li>
</ol>
3. Preparation of Monocytes
<ol>
	<li>Transfer HL-60 cells that are cultured in RPMI supplemented with 10% fetal calf serum, as suspension culture, into a 15ml tube. Centrifuge the cells at 200 x g for 5 min.</li>
	<li>Remove the supernatant and resuspend cells in 5 ml media and adjust the cell concentration with media to 2 million cells/ml. If fluorescence labeling of the HL-60 is desired, resuspend the cells in RPMI without serum and proceed as described in section 4.</li>
	<li>Add 0.5 ml of HL-60 cell suspension into each well of the 24 well cluster plate containing EC monolayer and incubate the plate in a cell culture incubator at 37 &#176;C and 5% carbon dioxide for a chosen fixed period of time between 1 and 3 hr. Little difference is observed between 1 hr and 3 hr time-point, where binding saturation is attained.</li>
	<li>Prepare for cell washing/harvesting: Fill a 120 ml tube with 100 ml of HBSS. Dispense 1 ml of formalin into each well of a fresh 24 well plate.</li>
	<li>After the incubation period, use a pair of sharp forceps and pick up the glass coverslip from the well and hold the coverslip vertical and dab the edge of the coverslip on a piece of tissue on the bench for 3 sec, taking care not to touch the cell-covered surface of the coverslip with the tissue.</li>
	<li>Holding the coverslip firmly with a pair of forceps, dunk the coverslip in and out of the HBSS five times.</li>
	<li>After the fifth dunk in HBSS, dab the edge of the coverslip on the tissue for 3 sec.</li>
	<li>Repeat the dunking and dabbing procedures described in 3.6 and 3.7, making a total of three sets of 5 dunks followed by a dab.</li>
	<li>Transfer the coverslip into a well containing 10% formalin to fix the cells at RT. The coverslip is ready for enumeration, for long-term storage or to be subjected to other procedures described below.</li>
</ol>
4. Labeling of HL-60 cells with Cell Tracker (optional)
<ol>
	<li>Count and transfer appropriate amount of HL-60 cells (e.g.,10 million) into a 15 ml centrifuge tube. Centrifuge HL-60 cells at 200 x g for 5 min. Remove supernatant.</li>
	<li>Resuspend cell pellet in Cell Tracker in RPMI media without serum at a concentration of 1 million cells/ml. Incubate cells in cell culture incubator for 1 hr.</li>
	<li>Centrifuge cells at 200 x g for 5 min and discard the supernatant. Resuspend cell pellet in media to a concentration of 2 million cells/ml.</li>
	<li>Add 0.5 ml of Cell Tracker-labeled HL-60 into each well containing endothelial cells grown on glass coverslip. Continue at 3.3.</li>
</ol>
5. Enumeration of Adhesive Endothelial Cells
<ol>
	<li>Mount coverslips on microscope slides with DAPI counter-stain to identify individual cells.</li>
	<li>Using an inverted microscope with a 4X or 10X objective, acquire images of several fields (e.g.,&#160;5 fields) and count the highest number of monocytes that aggregate on un-irradiated endothelial cells (This should typically be 3-5).</li>
	<li>Set twice this number as the threshold of monocytes on an endothelial cell to be that defined as a cluster. If required, a different criterion can be used for defining a cluster depending on the nature of the experiment.</li>
	<li>Count the number of clusters and the number of endothelial cells in the field. Divide the former by the latter to obtain the percentage of adhesive endothelial cells. Score numerous fields to obtain an average and standard deviation of the counts.</li>
</ol>
6. Counterstaining with Antibodies
<ol>
	<li>After the adhesion assay and cells on the coverslip have been fixed in 10% formalin for 15 min at RT, subject the coverslips to immunofluorescence using standard procedure10 and antibodies of choice.</li>
	<li>Add and remove the various solutions very gently to avoid dislodging monocytes that are attached to endothelial cells.</li>
</ol>
7. Counterstaining for Senescence-associated Beta-galactosidase Activity
<ol>
	<li>After the adhesion assay and cells on the coverslip have been fixed in formalin for 15 min, stain for senescence-associated beta-galactosidase activity as described in the instructions that accompany the staining kit. Add and remove the various solutions very gently to avoid dislodging monocytes that are attached to endothelial cells.</li>
</ol>

<ol>
	<li>Ortega-Gomez, A., Perretti, M., Soehnlein, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+of+inflammation:+an+integrated+view.">Resolution of inflammation: an integrated view.</a> EMBO Mol Med. 5 (5), 661-674 (2013).</li><li>Libby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+cardiovascular+disease+mechanisms.">Inflammation and cardiovascular disease mechanisms.</a> Am J Clin Nutr. 83 (2), 456S-460S (2006).</li><li>Ikuta, S., Kirby, J. A., Shenton, B. K., Givan, A. L., Lennard, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+endothelial+cells:+effect+of+TNF-alpha+on+peripheral+blood+mononuclear+cell+adhesion.">Human endothelial cells: effect of TNF-alpha on peripheral blood mononuclear cell adhesion.</a> Immunology. 73 (1), 71-76 (1991).</li><li>Watson, 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=IL-6+acts+on+endothelial+cells+to+preferentially+increase+their+adherence+for+lymphocytes.">IL-6 acts on endothelial cells to preferentially increase their adherence for lymphocytes.</a> Clin Exp Immunol. 105 (1), 112-119 (1996).</li><li>Sans, 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=VCAM-1+and+ICAM-1+mediate+leukocyte-endothelial+cell+adhesion+in+rat+experimental+colitis.">VCAM-1 and ICAM-1 mediate leukocyte-endothelial cell adhesion in rat experimental colitis.</a> Gastroenterology. 116 (4), 874-883 (1999).</li><li>Su, Y., Lei, X., Wu, L., Liu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+endothelial+cell+adhesion+molecules+P-selectin,+E-selectin+and+intercellular+adhesion+molecule-1+in+leucocyte+recruitment+induced+by+exogenous+methylglyoxal.">The role of endothelial cell adhesion molecules P-selectin, E-selectin and intercellular adhesion molecule-1 in leucocyte recruitment induced by exogenous methylglyoxal.</a> Immunology. 137 (1), 65-79 (2012).</li><li>Hallahan, D., Kuchibhotla, J., Wyble, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion+molecules+mediate+radiation-induced+leukocyte+adhesion+to+the+vascular+endothelium.">Cell adhesion molecules mediate radiation-induced leukocyte adhesion to the vascular endothelium.</a> Cancer Res. 56 (22), 5150-5155 (1996).</li><li>Vaporciyan, A. A., Jones, M. L., Ward, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+analysis+of+leukocyte-endothelial+adhesion.">Rapid analysis of leukocyte-endothelial adhesion.</a> J Immunol Methods. 159 (1-2), 93-100 (1993).</li><li>Prabhakarpandian, B., Shen, M. C., Pant, K., Kiani, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+for+modeling+cell-cell+and+particle-cell+interactions+in+the+microvasculature.">Microfluidic devices for modeling cell-cell and particle-cell interactions in the microvasculature.</a> Microvasc Res. 82, 210-220 (2011).</li><li>Lowe, D., Raj, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Premature+aging+induced+by+radiation+exhibits+pro-atherosclerotic+effects+mediated+by+epigenetic+activation+of+CD44+expression.">Premature aging induced by radiation exhibits pro-atherosclerotic effects mediated by epigenetic activation of CD44 expression.</a> Aging Cell. 13 (5), 900-910 (2014).</li></ol>

The authors have nothing to disclose.

The monocyte adhesion assay described above was used successfully in experiments designed to study the biological effects of ionizing radiation on endothelial monolayers10. Although this is not the only method available to assess the adhesiveness of an endothelial monolayer, it is the only method that allows the quantification of the proportion or percentage of endothelial cells within a monolayer that is adhesive. This is an important distinction as a global quantitative change in adhesion of monocytes, as measured by other methods can be attributed either to a general rise in adhesiveness of all cells of the endothelial monolayer or to the increase adhesiveness of a sub-population of endothelial cells within the monolayer, as shown in the example above. The value of having this information is exemplified by the fact that the ability to quantify the percentage of endothelial cells that exhibited enhanced adhesiveness after irradiation led to the inescapable conclusion that this effect is not due to genetic mutation of a particular gene as the percentage of cells that were adhesive were greatly above (over 1,000 times more) that which would be expected from random mutation of a gene by X-radiation at that dose.
The factor that allows good reproducibility of the results is the washing regime. As the washing procedure involves the immersion of the glass coverslip into the wash buffer followed by dabbing on a tissue, variability in the buffer turbulence which inevitably arises from the old method of pipetting of the wash buffer into a well is avoided. Indeed it was the observation of excessive variability between replicates obtained using the pipetting method that compelled us to devise a washing procedure that removed the variability element from the washing step.
Admittedly, the limitations of this assay lie in the need to carry out manual count of monocyte clusters, which do not allow it to be adapted for high throughput analyses. Secondly, the decision on how many monocytes constitute a cluster has to be determined semi-arbitrarily and the level may be set too high and result in the exclusion of some genuine clusters. The lack of shear force in this method can also be viewed as a limitation in experiments in which grossly enhanced adhesiveness of endothelial cells is induced (e.g., +TNF&#945;). In other situations however, the lack of shear force is an advantage as it allows the detection of less exaggerated augmentation of adhesiveness. Modest increase in monocyte adhesiveness can be particularly important and relevant. In vivo, monocytes would not (in a typical experimental time) be expected to attach in great numbers to endothelial cells with modest increase in adhesiveness, largely due to shear force. In time however, some monocytes probably would, and this is more likely to represent the environment of chronic inflammation. As such, the absence of shear force can be an advantage as it provides the opportunity for small increases in endothelial cell adhesiveness to be revealed in experimental time frames which are typically short and possibly too fleeting to allow modest increase in adhesiveness to be observed under shear stress.
The ability to subject the cells after this assay to other analyses such as senescence assay and immunofluorescence staining increases the usefulness of this method as it allows the association of adhesiveness of specific endothelial cells to particular cellular proteins or cellular states as demonstrated above, a feature that is not available with the older adhesion assays.
This method has been used with est2-immortalised human coronary endothelial cells and similar results were also obtained with primary human coronary endothelial cells10, demonstrating that it is not specific to just a particular cell line. Also, the adoption of this method of assaying adhesiveness of endothelial cells do not preclude subjecting the same cells to the standard adhesion assay that measures fluorescence of labeled immunocytes. Together, this report demonstrates that this method is versatile, inclusive and provides much more information than the standard adhesion assay.

Infiltration of immune cells such as monocytes across the endothelial cell layer that line blood vessels is an important step in the process of inflammation1. This allows the homing of immune cells (immunocytes) to a site of injury. In other instances and locations such as the coronary artery and carotid artery, infiltration of monocytes through the endothelial layer can lead to the undesirable long term residence of these cells in the wall of the artery, potentially leading to the formation of plaques2. In all cases of immunocyte infiltration, the first step involves the activation of the endothelial cells in a localized region of the blood vessel. Endothelial cells are activated by pro-inflammatory cytokines such as TNF-&#945; and IL-6 to increase expression of cell surface proteins such as VCAM, ICAM and E-selectin which facilitate the recruitment and attachment of the immunocytes onto the endothelial cell surface3-6.
Initially, measurement of endothelial cell adhesion was carried out by counting monocytes that adhered to endothelial monolayer 7. The limitations of this method in terms of precision led to the use of radio-labeled lymphocyte or monocyte, followed by quantification of radioactive material which corresponds to lymphocytes or monocytes adhesion 4. This method was eventually superseded by a fluorescence-based method, whereby immunocytes of interest were labeled with fluorescence dye and subjected to the same procedure as above with the difference being that fluorescence instead of radioactivity is measured 8. Presently this method has emerged as the most convenient and is sold in kit form by several commercial suppliers. While this assay can measure relative levels of adhesiveness between controls and experimental samples, it does not reveal whether a change in fluorescence is due to a uniform change in adhesiveness across the entire endothelial cell population or if the change is due to a difference of adhesiveness within a sub-population of cells. It is also evident that the stringency of washing exerts a profound effect on the result, and more importantly, the uniformity of rinsing off unbound monocytes between different endothelial cell monolayers will impact greatly on the closeness of results from replicates and reproducibility of the results between samples. More recently, several systems that pump monocytes in media across an endothelial cell monolayer were used to address this problem 9. In addition, these flow systems also recapitulate the effect of shear force on the endothelial cells. While the advantages of such systems are clear and very attractive, it is also important to realize that while endothelial cell adhesiveness can be greatly augmented, as is the case in acute inflammation, by factors such as TNF&#945;, some other activators, such as ionizing radiation10 elicit changes that are not readily detected within in vitro experimental time frames by these very stringent systems. While such minute changes of endothelial cell adhesiveness are easily missed or dismissed in vitro, it is not necessarily benign in vivo, where such small changes within a life time, which is characteristic of chronic inflammation, can exert very significant outcomes. Hence a robust, yet sensitive, and specific method to detect and measure endothelial cell adhesiveness is needed.
Here, we report a method to measure adhesiveness of endothelial cells directly. This method does not rely on fluorescence measurement as an indirect surrogate indicator of endothelial cell adhesiveness. It reveals whether changes in adhesiveness are due to uniform change across all cells or confined to a sub-population. Furthermore, it allows co-staining of endothelial cells with markers such as senescence-associated beta-galactosidase, cell viability marker, Calcien AM and antibodies against cell surface and intracellular proteins, allowing the association of individual adhesive endothelial cells to certain cell states or expression of specific proteins.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Hepes Buffered Saline Solution (HBSS)</td>
			<td>Sigma</td>
			<td>H6648</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips Round 12mm Diameter</td>
			<td>Menzel-Glaser</td>
			<td>CB00120RA1</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well cluster plate</td>
			<td>Costar</td>
			<td>3526</td>
			<td></td>
		</tr>
		<tr>
			<td>Meso-Endo Cell media</td>
			<td>Cell Applications</td>
			<td>212-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma</td>
			<td>T4174</td>
			<td></td>
		</tr>
		<tr>
			<td>Soybean trypsin inhibitor</td>
			<td>Life Technologies</td>
			<td>17075-029</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Tracker Green</td>
			<td>Life Technologies</td>
			<td>C7025</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Sigma</td>
			<td>R8758</td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal Calf Serum</td>
			<td>Life Technologies</td>
			<td>10500064</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta glasctosidase Assay kit&#160;&#160;</td>
			<td>CellSignaling</td>
			<td>9860</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma</td>
			<td>F0895</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitation of endothelial cell adhesiveness in vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

This method allows the detection of individual adhesive endothelial cells within a population. For example, while a monolayer of un-irradiated endothelial cells retained a few sporadic HL-60 monocytes following incubation and washing, endothelial monolayer at 7 days post-10Gy irradiation prior to incubation with monocytes were bound by monocytes in clusters around individual endothelial cells (Figure 1). Although this phenomenon is readily observable under phase contrast microscopy, fluorescence microscopy reveals an even clearer image of the monocyte clusters.
After performing the described adhesion assay, it is possible to proceed to staining the cells for membrane, cytoplasmic or nuclear (Figure 2) proteins using appropriate antibodies with standard immunofluorescence methods. Furthermore, enzyme-based assay such as senescence-associated beta-galactosidase (Figure 3) can also be performed.
Since each monocyte cluster corresponds to an individual endothelial cell, enumeration of the clusters will reveal the actual number of adhesive endothelial cells within the monolayer (Figure 4), and hence the percentage of adhesive endothelial cells within the population (Table 1, Figure 6). This is the only method to date that allows such quantification of endothelial cell adhesiveness. It is noteworthy that the endothelial cells used in the experiments here are contact-inhibited and do not increase in number after achieving confluence. This eliminates the potential complexity posed by increased endothelial cell number at later time points. If desired, monocytes attached to endothelial monolayers can be dislodged by 0.5% trypsin-0.2% EDTA solution (instead of fixation in 3.9) and their fluorescence measured using a plate reader, as is the case in contemporary methods (Figure 5).
<img alt="Figure 1" src="/files/ftp_upload/52924/52924fig1.jpg" /> 
Figure 1.&#160;Adhesion of monocytes on monolayer of endothelial cells. On un-irradiated endothelial cells, monocytes adhered as individual cells in a sporadic and random manner (left panels) and in clusters on individual endothelial cells of the 7 days post-10 Gy irradiated monolayer (right panels). Lower panels are fluorescence images of the top panels, confirming that the small and bright spherical cells visible under phase contrast are indeed monocytes that were pre-labeled with Cell-Tracker Green. Some of the monocyte clusters are indicated by white arrows. Images were taken using a 10X objective. <a href="https://www.jove.com/files/ftp_upload/52924/52924fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52924/52924fig2.jpg" /> 
Figure 2.&#160;Staining of endothelial cells for proteins post-adhesion assay. After performing the described adhesion assay, cells on the glass coverslips were subjected to immunofluorescence for the detection of (A) membrane protein (CD44), b) cytoplasmic protein (Tubulin) or c) nuclear protein (&#947;-H2AX). Left panels of (A) and (B) (seen with 20X objective) show monocytes pre-labelled with Cell Tracker Green and the similar image on the right panels reveal CD44 and microtubules (red) and DAPI-stained nuclei (blue). White arrows in (C) point to irradiated endothelial cell nuclei that were stained with antibodies against &#947;-H2AX. Monocyte nuclei which are stained a brighter blue by DAPI are easily distinguished from the oval shaped nuclei of the endothelial cells. Images were taken with 20X objective. <a href="https://www.jove.com/files/ftp_upload/52924/52924fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52924/52924fig3.jpg" /> 
Figure 3.&#160;Senescence-associated beta-galactosidase staining of endothelial cells. After adhesion assay, cells on coverslips were subjected to staining for senescence-associated beta-galactosidase which causes lysosomes of senescent cells to turn blue, as is evident in the endothelial cell that is selectively bound by numerous monocytes, which are easily identified due to their small size and spherical shape. Image seen through 20X objective. <a href="https://www.jove.com/files/ftp_upload/52924/52924fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52924/52924fig4.jpg" /> 
Figure 4. Enumeration of monocyte clusters on individual endothelial cells. After adhesion assay, images of the cells were taken from several different positions and monocyte clusters identified and circled (in green). A cluster was defined as an agglomeration of 10 or more monocytes on an endothelial cell. The number of monocyte clusters and number of 12 days post-irradiated endothelial cells (Dotted in red) in the image were counted and percentage of adherent endothelial cells calculated as shown in Table 1. Image taken through 10X objective. <a href="https://www.jove.com/files/ftp_upload/52924/52924fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52924/52924fig5.jpg" /> 
Figure 5. Quantification of fluorescence of adherent monocytes. Following adhesion assay, cells on glass coverslips were trypsinised and the fluorescence of monocytes pre-labelled with CellTracker Green was measured, using a fluorescence plate reader, as an indirect indicator of the adhesiveness of the endothelial monolayer. Results from such measurements at stated days post-irradiation were obtained and platted on the graph above.
<img alt="Figure 6" src="/files/ftp_upload/52924/52924fig6.jpg" /> 
Figure 6. Representation of adhesion assay scores from Table 1. Adhesive endothelial cells in five different locations on three glass coverslips were scored as described in Figure 4 and the results tabulated above demonstrating that 12 days after 10 Gy irradiation, 8 to 10 percent of irradiated endothelial cells became adhesive.
<table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="3">Percentage of adhesive 
			endothelial cells</td>
		</tr>
		<tr>
			<td></td>
			<td>Disc 1</td>
			<td>Disc 2</td>
			<td>Disc 3</td>
		</tr>
		<tr>
			<td>Field 1</td>
			<td>8.65</td>
			<td>8.29</td>
			<td>8.21</td>
		</tr>
		<tr>
			<td>Field 2</td>
			<td>10.44</td>
			<td>7.27</td>
			<td>7.21</td>
		</tr>
		<tr>
			<td>Field 3</td>
			<td>9.63</td>
			<td>9.05</td>
			<td>8.33</td>
		</tr>
		<tr>
			<td>Field 4</td>
			<td>11.11</td>
			<td>7.09</td>
			<td>8.41</td>
		</tr>
		<tr>
			<td>Field 5</td>
			<td>10.55</td>
			<td>9.4</td>
			<td>11.61</td>
		</tr>
		<tr>
			<td>Average</td>
			<td>10.07</td>
			<td>8.22</td>
			<td>8.75</td>
		</tr>
	</tbody>
</table>
Table 1. Adhesion assay scores. Adhesive endothelial cells in five different locations on three glass coverslips were scored as described in Figure 4 and the results tabulated above demonstrating that 12 days after 10 Gy irradiation, 8 to 10 percent of irradiated endothelial cells became adhesive.

Watch this Scientific Journal Video about Quantitation of Endothelial Cell Adhesiveness In Vitro at JoVE.com

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

quantitation-of-endothelial-cell-adhesiveness-in-vitro

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Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &amp;#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.	
Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.

Measuring biomarkers in complex biological samples is increasingly guiding clinical decision-making. We describe a highly sensitive method to simultaneously measure cardiac myosin binding protein-C, creatine kinase MB, and cardiac troponin I in serum samples from subjects with myocardial infarction and healthy control subjects.

Biomarkers are becoming increasingly more  important in clinical decision-making, as well as basic science. Diagnosing  myocardial infarction (MI) is ...

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...