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><tbody><tr><td>Hepes Buffered Saline Solution (HBSS)</td><td>Sigma</td><td>H6648</td><td></td></tr><tr><td>Glass coverslips Round 12 mm 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&amp;nbsp;&amp;nbsp;</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.

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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 ...

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11.5K Views.  Centre for Radiation, Chemicals and Environmental Hazards, Public Health England. We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer. 

Video: Quantitation of Endothelial Cell Adhesiveness In Vitro

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

Bioengineering

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

Immunology and Infection

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days.

Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions ...

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

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

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

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

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Dynamic Adhesion Assay for the Functional Analysis of Anti-adhesion Therapies in Inflammatory Bowel Disease

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a crucial step in this process and therapeutic strategies interfering with adhesion have been successfully established. The anti-&#945;4&#946;7 integrin antibody, vedolizumab, is used for the clinical treatment of Crohn&#39;s disease (CD) and ulcerative colitis (UC) and further compounds are likely to follow.
The details of the adhesion procedure and the action mechanisms of anti-integrin antibodies are still unclear in many regards due to the limited available techniques for the functional research in this field.
Here, we present a dynamic adhesion assay for the functional analysis of human cell adhesion under flow conditions and the impact of anti-integrin therapies in the context of IBD. It is based on the perfusion of primary human cells through addressin-coated ultrathin glass capillaries with real-time microscopic analysis. The assay offers a variety of opportunities for refinements and modifications and holds potentials for mechanistic discoveries and translational applications.

Dynamic adhesion of immune cells to the vessel wall is a prerequisite for gut homing. Here, we present a protocol for a functional in vitro assay for the impact analysis of anti-integrin antibodies, chemokines or other factors on the dynamic cell adhesion of human cells using addressin-coated capillaries.

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

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