This work was supported by the Ministerio de Econom&#237;a y Competitividad (MINECO) and the Instituto de Salud Carlos III (ISCIII) (grant number IERPY 1149/16 to A.L.; MPY 1410/09 to S. Hortelano); by the MINECO through the Fondo de Investigaci&#243;n en Salud (FIS) (grants numbers PI11.0036 and PI14.0055 to S. Hortelano). S. Herranz was supported by IERPY 1149/16 from ISCIII.

1. Endothelial Cell Culture
<ol>
	<li>Tissue culture treated plates
	<ol>
		<li>Coat 100 mm tissue culture plates with 2.5 mL gelatin solution (autoclaved, 0.1% gelatin in distilled water) for 30 min at 37 &#176;C; this can be extrapolated to the required well format. Aspirate the gelatin solution and leave plates to air-dry in the tissue culture hood.</li>
	</ol>
	</li>
	<li>Tissue culture conditions
	<ol>
		<li>Cultivate the MLEC-04 cells inside a biological incubator (37 &#176;C, 95% humidity, 5% CO2). Grow the cells in complete media of Dulbecco&#39;s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12) supplemented with 10% Fetal Bovine Serum (FBS) and 100 units/mL penicillin and 100 &#181;g/mL streptomycin (P/S).</li>
		<li>Count the cells by the standard Neubauer chamber method, and add the MLEC-04 cells to the 100 mm gelatin-treated plates in 10 mL complete media, at a low density of 2 - 11 x 104 cells/cm2. Split the cells 1:3 when they reach confluence. 
		NOTE: Usually this occurs after two to three days in culture.</li>
		<li>Subculture the cells by washing the plates with 10 mL sterile Phosphate Buffered Saline (PBS) followed by adding 2 mL trypsin-EDTA solution (0.25% trypsin, 5 mM EDTA) and incubate for 3 min at 37 &#176;C.</li>
		<li>Stop the trypsin-EDTA reaction and recover the suspended cells by adding 10 mL complete media. Spin down the cells (300 x g, 5 min), discard the supernatant, resuspend the cellular pellet in complete media and subculture appropriately.</li>
	</ol>
	</li>
</ol>
2. LPS Treatment and Mediators
<ol>
	<li>Plate the MLEC-04 cells in complete media at sub-confluence into the following well format: 7 x 105 cells/well on 6-well plates and 2.5 x 105 cells/well on 96-well plates.</li>
	<li>Incubate the culture for 6 h in a cell incubator (37 &#176;C, 95% humidity, 5% CO2). Switch the cells to incomplete media (DMEM-F12, P/S) and leave overnight (ON) to synchronize and to reduce the cell activity.</li>
	<li>Remove the media and test novel pharmacological compounds by incubating the endothelial cells with (or without) the drug in question (e.g. DT10) diluted in the incomplete media (30 min, 37 &#176;C); add 1.4 mL/well for 6-well plate or 0.14 mL/well for 96-well plates).</li>
	<li>Challenge the cells by adding LPS to the incubation media (100 ng/mL, final concentration) for the period of time specified in each assay. Use PBS as a control.</li>
</ol>
3. Evaluation of Transcriptional Profile on Activated Endothelium by RT-qPCR
<ol>
	<li>Seed the MLEC-04 cells at sub-confluence in 6-well culture plates (7 x 105 cells/well). Incubate for 6 h (37 &#176;C, 95% humidity, 5% CO2) and afterwards proceed to serum starvation (incubate ON).</li>
	<li>Remove the media and treat (or do not treat) the endothelial cell cultures with the drug of interest diluted in the incomplete media (incubate 30 min, 37 &#176;C); add 1.4 mL/well for 6-well plate (30 min, 37 &#176;C).</li>
	<li>Challenge the cells by adding 100 ng/mL LPS to the incubated media (as described in step 2.3) and incubate for 6 h. Stop the reaction by washing twice with cold PBS and keep plates at -80 &#176;C until sample processing.</li>
	<li>RNA extraction
	<ol>
		<li>Thaw the plates and add 1 mL/well RNA extraction buffer (38% phenol and 0.8 M guanidinium isothiocyanate; see table of materials). Leave for 30 min at room temperature (RT) under agitation and collect homogenate in 1.5 mL tubes.</li>
		<li>Add 200 &#181;L chloroform and agitate gently for 15 s. Incubate for 3 min at RT and centrifuge (11,600 x g, 15 min, 4 &#176;C).</li>
		<li>Transfer aqueous phase to another 1.5 mL tube. Precipitate RNA by adding 500 &#181;L isopropanol followed by a 10 min incubation at RT and then centrifugation (11,600 x g, 4 &#176;C).</li>
		<li>Discard the supernatant and wash the pellet with 75% ethanol by vortex agitation and then centrifugation (7,500 x g, 4 &#176;C).</li>
		<li>Air-dry the pellet and solubilize RNA in 25 &#181;L pure H2O by incubating for 10 min at 55 &#176;C. Keep RNA at -80 &#176;C until sample processing.</li>
	</ol>
	</li>
	<li>Quantity and purity of RNA
	<ol>
		<li>Quantify the RNA concentration by measuring the absorbance of the sample at 260 nm in a spectrophotometer (see Table of Materials).</li>
		<li>Measure at 230 nm and 280 nm to determine the RNA purity. 
		NOTE: The ratio at 260/280 indicates protein or phenol contamination. A ratio close to 2 is acceptable. The ratio at 260/230 indicates EDTA, carbohydrates or phenol contaminants. Values between 2.0 - 2.2 are acceptable.</li>
	</ol>
	</li>
	<li>Checking RNA integrity
	<ol>
		<li>Run 2 &#181;g of total RNA and an RNA ladder on a 1.5% denaturing agarose gel; visualize on a gel documentation system (see Table of Materials). 
		NOTE: A 2:1 ratio of clear and sharp 28S and 18S rRNA bands indicates an intact RNA. Partially degraded RNA resolves as a smeared appearance.</li>
	</ol>
	</li>
	<li>RT-qPCR
	<ol>
		<li>Synthesize the complementary DNA (cDNA) from a single stranded RNA following the standard protocol (see Table of Materials)11.</li>
	</ol>
	</li>
	<li>Evaluate gene expression by RT-qPCR
	<ol>
		<li>Prepare the reaction mixture for each sample: Mix 2 &#181;L cDNA, 7 &#181;L fluorescent compound, 300 nM forward primer, and 300 nM reverse primer (Table 1) in a final volume of 13 &#181;L, and add to the 96-Well Reaction Plate (see Table of Materials).</li>
		<li>Seal the plate with an optical adhesive cover, centrifuge (300 x g, 1 min) and run the reaction on a Real-Time PCR System (hot start 95 &#176;C for 20 s followed by 40 cycles: 95 &#176;C for 3 s and 60 &#176;C for 30 s) (see Table of Materials).</li>
		<li>Analyze the results by relative quantitation using the comparative method 2-&#916;&#916;Ct with the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or acidic ribosomal phosphoprotein P0 (36B4)12.</li>
	</ol>
	</li>
</ol>
4. Assess Endothelial Activation by Flow Cytometry
<ol>
	<li>Treat the MLEC-04 cells following the conditions described in section 2 and evaluate the changes in the endothelial surface proteins by flow cytometry.</li>
	<li>Detach the cells after 6 h of LPS stimulation with the trypsin-EDTA method described in step 1.2.3, and wash with the incomplete media at 4 &#176;C.</li>
	<li>Count the cells using the Neubauer chamber method and place 10 x 104 cells into u-bottom 96-well plates. Centrifuge (300 x g, 5 min) and discard supernatant by a 180&#176; snap of the wrist from top to the bottom and quick recovery to the original position.</li>
	<li>Add 50 &#181;L of the selected antibody diluted in the incomplete media (10 &#181;g/mL, final concentration) to the cells and incubate (30 min, 4 &#176;C).</li>
	<li>Wash the cells twice with the incomplete media following the procedure described in step 4.3, and incubate with 50 &#181;L at 10 &#181;g/mL of the corresponding secondary antibody coupled to FITC or a similar conjugate (30 min, 4 &#176;C in a dark space).</li>
	<li>Wash the cells once with the incomplete media followed by a PBS wash. Recover the cells with 300 &#181;L PBS using 1 mL pipette tips and place in cytometry tubes.</li>
	<li>Evaluate the samples in the flow cytometry system (see Table of Materials). Adjust the cell population by the forward and side scattering parameters. Gate the population of interest. Adjust gates based on the negative control from cells incubated with isotype control. Analyze the results as the percentage of positive cells or mean fluorescence intensity8.</li>
</ol>
5. Evaluate Endothelial Activation by Western Blot
<ol>
	<li>Seed the endothelium at sub-confluence in 6-well culture plates (7 x 105 cells/well) and treat with LPS as described in section 2. Analyze the inflammatory protein expression by the following western blot protocol.</li>
	<li>Stop the LPS stimulation at different time points to elucidate different aspects of the endothelial response:
	<ol>
		<li>Ascertain the inflammatory proteins profile after 6 h of LPS stimulation.</li>
		<li>Determine the cell signaling every 15 min through a 60 min period.</li>
	</ol>
	</li>
	<li>In both cases, stop the reaction by washing twice with cold PBS and store the samples at -80 &#176;C until sample processing.</li>
	<li>Lyse cells and protein extraction
	<ol>
		<li>Add 200 &#181;L lysis buffer to each well (1% non-ionic surfactant, 10 mM Tris-HCl, 1mM EDTA, 150 mM sodium chloride, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 2.1 mM sodium orthovanadate at pH 7.6, supplemented with protease inhibitor cocktail) (see table of materials).</li>
		<li>Incubate for 15 min at 4 &#176;C under agitation, scrape wells with a pipette tip and collect the homogenate in 1.5 mL tubes.</li>
		<li>Centrifuge the tubes at 11,600 x g at 4 &#176;C.</li>
		<li>Store the supernatant in clean 1.5 mL tubes at -80 &#176;C until processing.</li>
	</ol>
	</li>
	<li>Measure the protein concentration by the bicinchoninic acid assay following the standard protocol (see table of materials)13.</li>
	<li>Resolve the 30 &#181;g of total protein lysate for each sample by the standard technique of 0.1% sodium dodecyl sulfate, 10% polyacrylamide gel electrophoresis (SDS-PAGE)14. Run samples with 10 mM &#946;-mercaptoethanol (reducing conditions) or without (non-reducing conditions) depending on the antibody used to detect the protein of interest.</li>
	<li>Transfer the separated proteins from the polyacrylamide gel to a polyvinylidene difluoride (PVDF) transfer membrane with 0.45 &#181;m pore size using standard procedure.</li>
	<li>After transference, wash the membrane twice with PBS, 0.1% polysorbate-20 (PBS-T) and block unspecific binding sites by adding 20 mL 2% BSA diluted in PBS-T for detected phosphoproteins or 5% non-fat dry milk in PBS-T for total proteins, under agitation (90 min, RT).</li>
	<li>Dilute the selected antibody in 10 mL of the appropriate blocking buffer at the recommended concentration and incubate the membrane under agitation (ON, 4 &#176;C). Alternatively, place the membrane in sealed plastic bags and used 5 mL of the diluted antibody.</li>
	<li>Next day, wash the membrane three times (excess PBS-T, 15 min, RT).</li>
	<li>Incubate the membrane under agitation (30 min, RT) with 10 mL of the correspondent secondary antibody bound to horseradish peroxidase (HRP) diluted at 1:10,000 in the appropriate blocking buffer.</li>
	<li>Wash the membrane three times under agitation (excess PBS-T, 15 min, RT).</li>
	<li>Incubate the membrane for 1 min with 0.1 mL/cm2 of the peroxidase substrate enhanced chemiluminescence (ECL). Place the drained membrane between two transparent plastic sheets, and insert it into the chemiluminescence detection system which includes a Charged-Coupled Device camera (CCD).
	<ol>
		<li>Use the CCD camera to detect the signal and its software to record images with the accumulative program (images display accumulated signal at every 30 s exposure for a total 15 min period).</li>
	</ol>
	</li>
	<li>Quantify the band intensity by densitometry using the ImageJ software following the protocol described in the user guide15.
	<ol>
		<li>Open the sample in the ImageJ software and select the band of interest with rectangular selection tool.</li>
		<li>Select as the first lane and press plot lanes to obtain the profile plots.</li>
		<li>Delimit the area of the peaks with the straight-line selection.</li>
		<li>Measure the size of each band by clicking the inside of each peak.</li>
		<li>Represent the results with respect to loading protein control (&#946;-actin, tubulin, etc.).</li>
	</ol>
	</li>
</ol>
6. Evaluate Endothelial Factor Released from Leukocyte Activation by Adhesion Assays
<ol>
	<li>Obtain the endothelial conditioned media.
	<ol>
		<li>Treat the MLEC-04 cells as indicated in section 2 and collect the culture supernatant after 24 h stimulation with LPS (100 ng/mL).</li>
		<li>Collect the conditioned media from the treated MLEC-04 cells and centrifuge (600 x g). Keep the supernatant in 0.5 mL aliquots at -80 &#176;C until use.</li>
	</ol>
	</li>
	<li>Leukocyte adhesion assay
	<ol>
		<li>Coat the 96-well plates with 50 &#181;L/well of selected extracellular matrix proteins diluted in PBS (except for collagen, which is diluted in 0.1 M acetic acid): fibronectin (1 - 10 &#181;g/mL), laminin (1 - 10 &#181;g/mL) and collagen type I (10 - 40 &#181;g/mL). Leave ON at 4 &#176;C.</li>
		<li>Discard the coated media and block unspecific binding sites on wells with 150 &#181;L PBS, 1 % BSA heat-inactivated (1 min, 100 &#176;C) for 90 min at RT.</li>
		<li>Wash the wells twice with PBS and add 100 &#181;L of previously collected endothelial conditioned media (section 6.1) to the wells. 
		NOTE: Plates are ready for performing the adhesion assay.</li>
		<li>Culture the mouse monocyte-macrophage cell line J774 in a biological incubator (37 &#176;C, 95% humidity, 5% CO2) with Roswell Park Memorial Institute Medium (RPMI), 10% FBS, 1% P/S. 
		NOTE: The J774 cells grow in suspension.</li>
		<li>Collect the J774 cells into a 15 mL tube and centrifuge (300 x g, 5 min). Discard the supernatant and resuspend the cellular pellet with 10 mL serum-free media. Count the cells in a Neubauer chamber. Centrifuge the cells (300 x g, 5 min), discard the supernatant and resuspend the cells in serum-free media at 15 x 104 J774 cells per 50 &#181;L media.</li>
		<li>Add 15 x 104 J774 cells to each well in 50 &#181;L serum-free media and incubate for 15 min at 4 &#176;C followed by 1 h at 37 &#176;C in the CO2 cell incubator.</li>
		<li>Wash the wells twice, gently by adding warm PBS; centrifuge (300 x g, 5 min) and discard supernatant by a 180&#176; snap of the wrist from top to the bottom and quick recovery to the original position. Fix the attached cells by adding 100 &#181;L/well of 4% paraformaldehyde (PFA) in PBS and incubate (10 min, RT).</li>
		<li>Discard the media gently and permeabilize the cells with 2% methanol in PBS for 2 min at RT. Discard the media gently and stain the cells by adding 50 &#181;L/well of 0.5% crystal violet in 20% methanol for 90 s at RT.</li>
		<li>Wash the plate with abundant running water, discard the excess liquid and leave it to air-dry. Report the attached cells by digital camera coupled to a light microscope.</li>
		<li>Dilute the cell staining by adding 100 &#181;L/well of 0.1 M sodium citrate, 50% ethanol. Measure the absorbance at 545 nm and represent the results as arbitrary units of absorbance or percentage of adhesion by considering 100% as cell adhesion reached to wells coated with higher ligand concentration</li>
	</ol>
	</li>
</ol>
7. Test Endothelial Activation by Leukocyte-Endothelium Co-adhesion Assay
<ol>
	<li>Treat the MLEC-04 cells on 96-well plates as described in section 2 and incubate with LPS (6 h, 37 &#176;C).</li>
	<li>Fluorescently label J774 cells with Carboxyfluorescein Succinimidyl Ester (CSFE).
	<ol>
		<li>Wash the J774 cells in PBS following the procedure in 6.2.5. Count the cells by the Neubauer chamber method and resuspend at 1 x 106 cells/mL in PBS, 0.1% BSA.</li>
		<li>Incubate the cells with CFSE (5 &#181;M, final concentration) for 20 min at 37 &#176;C. Add 5 mL incomplete media and incubate (5 min, 4 &#176;C).</li>
		<li>Wash once with incomplete media as explained in 6.2.5., resuspend at 15 x 104 cells/100 &#181;L and proceed to the co-adhesion assay.</li>
	</ol>
	</li>
	<li>After the endothelium treatment, wash the wells three times with incomplete media. Add CFSE-J774 cells (15 x 104 cells/100 &#181;L) to each endothelial-coated well. Incubate the plate for 10 min at 4 &#176;C followed by 60 min at 37 &#176;C.</li>
	<li>Wash the wells gently, as described in 6.2.7., using warmed PBS and report the J774 adhesion to the endothelial layer by the following two methods:
	<ol>
		<li>Lyse the cells in 0.1 M Tris-HCl pH 8.8, 1% SDS (100 &#181;L/well) and measure the CFSE-J774 signal by fluorometry (excitation/emission = 492 nm/517 nm). Represent the results as arbitrary units of fluorescence intensity or percentage of adhesion with respect to positive control (signal from total cells added to each well).</li>
		<li>Fix the cells in 4% PFA and report the attachment of the CFSE-J774 cells to the endothelium by visualizing under a fluorescence microscope (excitation/emission = 492 nm/517 nm).</li>
	</ol>
	</li>
</ol>

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</ol>

The authors have nothing to disclose.

This endothelial protocol describes a stepwise technology that establishes the groundwork for exploring novel mechanisms involved in the regulation of the inflammatory response. These approaches are based on the study of the endothelial activity stimulated by LPS and evaluate the critical steps involved in leukocyte recruitment during the inflammatory response, specifically: endothelial cytokines release, endothelial adhesion molecules expression and leukocyte adhesion to the vascular layer. Once the endothelial paramete...

Inflammation is a beneficial biological response against infectious agents, with the major aim to eliminate the pathogen and repair damaged tissue. Under certain conditions, such as chronic infections or autoimmune diseases, inflammation does not resolve. Instead, there is an aberrant reaction with continuous infiltration of leukocytes, resulting in a prolonged immune response that leads to tissue damage, fibrosis, loss of function, and overall, disability and in some cases death of the patient. These human disorders, cataloged as inflammatory diseases, all involve the blood vessels for the control of leukocyte extravasation1,2.
The endothelial cells play a fundamental role in the regulation of the inflammatory response by controlling leukocyte trafficking. When the endothelial layer is exposed to inflammatory mediators such as LPS, the resting endothelium activates and expresses pro-inflammatory cytokines (Cxcl10, Cxcl5, Cxcl1, etc.) and adhesion molecules (E-selectin, VCAM-1 and ICAM-1) that favor recruitment of circulating leukocytes to the infection site. The leukocytes primed by the released cytokines then mediate rolling and interaction with the endothelial layer through the correspondent adhesive counterparts: PSGL-1 to selectin, &#945;4&#946;1 integrin to VCAM-1, and &#945;L&#946;2 integrin to ICAM-1. Finally, the leukocytes migrate across the vasculature towards the focus of inflammation3.
The essential role of the endothelium in regulating the inflammatory response has been demonstrated on mice that were genetically modified to express the LPS receptor, toll-like receptor 4 (TLR4), only on the endothelial cells. These endothelial-TLR4 animals were able to respond to an LPS-mediated inflammation and to detect the infection generated after bacteria inoculation, and consequently achieve infection resolution and survival at similar levels as the wild type mice4,5.
For the endothelium-regulated inflammatory response pathway, it has been postulated that the inhibition at some stages of the leukocyte-endothelium interaction would result in the reduction of trans-endothelial migration and a better prognosis for inflammatory-related diseases. In fact, several strategies targeting the endothelial activation and leukocyte-endothelium interaction have been designed to hinder extravasation of immune cells as a treatment for inflammatory disorders6,7.
In this report, we describe a thorough group of in vitro techniques to fully characterize the endothelial activity in response to the inflammatory stimulus LPS and its role in leukocyte activation and adhesion to the vascular layer. The endothelial cell model used in this manuscript was the mouse lung endothelial cell line (MLEC-04), as described by Hortelano et al.8. The MLEC-04 cell line has been validated in the literature to be an appropriate system to study endothelial activation9,10. Based on research interests, these approaches can be easily extrapolated to any endothelial or leukocyte systems and inflammatory profile. Once the endothelial parameters in the selected conditions are defined, the system can test novel drugs on the proposed experimentation to evaluate the vascular activation. In this inflammatory context, the endothelium cells tested with the compound of interest can be compared to the control conditions of the cells, and any resulting differences may inform the drug&#39;s prognostic outcome on development and progression of inflammation. To conclude, we propose a relevant system to characterize new drug targets to the endothelial cells, which can influence the design of novel vascular-specific therapies against inflammatory-related diseases.

<table><tbody><tr><td>Gelatin</td><td>Sigma</td><td>G9391</td><td></td></tr><tr><td>DMEM-F12</td><td>Lonza</td><td>BE12-719F</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma</td><td>A4503</td><td></td></tr><tr><td>Penicillin streptomycin</td><td>Lonza</td><td>DE17-602E</td><td></td></tr><tr><td>Trypsine</td><td>Lonza</td><td>BE17-160E</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>ED2SS</td><td></td></tr><tr><td>LPS</td><td>Sigma</td><td>L2880</td><td></td></tr><tr><td>Trizol</td><td>Sigma</td><td>T9424</td><td>RNA extraction buffer</td></tr><tr><td>Isopropanol</td><td>Sigma</td><td>33539</td><td></td></tr><tr><td>Ethanol absoluto</td><td>Panreac</td><td>1,310,861,612</td><td></td></tr><tr><td>Pure H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Qiagen</td><td>1017979</td><td>RNAse free</td></tr><tr><td>Agarose</td><td>Pronadisa</td><td>8020</td><td></td></tr><tr><td>Stain for agarose gels</td><td>Invitrogen</td><td>s33102</td><td></td></tr><tr><td>SuperScript III First-Strand Synth</td><td>Invitrogen</td><td>18080051</td><td>Reagents for RT-PCR</td></tr><tr><td>Fast SYBR Green Master Mix</td><td>Applied Biosystems</td><td>4385610</td><td>Fluorescent stain for qPCR</td></tr><tr><td>MicroAmp Fast Optical 96-Well</td><td>Applied Biosystems</td><td>4346906</td><td>Plates for qPCR</td></tr><tr><td>U-bottom 96 well plates</td><td>Falcon</td><td>353072</td><td></td></tr><tr><td>Cytometry tubes</td><td>Falcon</td><td>352054</td><td></td></tr><tr><td>TX100</td><td>Panreac</td><td>212314</td><td>Non-ionic surfactant</td></tr><tr><td>Tris-HCl</td><td>Panreac</td><td>1,319,401,211</td><td></td></tr><tr><td>Sodium chloride</td><td>Merck</td><td>1,064,041,000</td><td></td></tr><tr><td>Sodium pyrophosphate</td><td>Sigma</td><td>221368</td><td></td></tr><tr><td>Sodium fluoride</td><td>Sigma</td><td>S7920</td><td></td></tr><tr><td>Sodium orthovanadate</td><td>sigma</td><td>13721-39-6</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>sigma</td><td>P8340</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Pierce</td><td>23225</td><td>Reagents for bicinchoninic acid assay</td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>merck</td><td>805,740</td><td></td></tr><tr><td>PVDF Transfer Membrane, 0.45 &amp;micro;m</td><td>Thermo Scientific</td><td>88518</td><td></td></tr><tr><td>Tween-20</td><td>Panreac</td><td>1,623,121,611</td><td>Polysorbate 20</td></tr><tr><td>PBS</td><td>Lonza</td><td>BE17-515Q</td><td></td></tr><tr><td>ECL</td><td>Millipore</td><td>WBKLS0500</td><td></td></tr><tr><td>Fibronectin</td><td>Sigma</td><td>F1141</td><td></td></tr><tr><td>Laminin</td><td>Sigma</td><td>L2020</td><td></td></tr><tr><td>Collagen type I</td><td>Sigma</td><td>c8919</td><td></td></tr><tr><td>Acetic acid</td><td>Panreac</td><td>1,310,081,611</td><td></td></tr><tr><td>Trypan blue</td><td>Sigma</td><td>T8154</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Methanol</td><td>Panreac</td><td>1,310,911,612</td><td></td></tr><tr><td>Crystal violet</td><td>Sigma</td><td>HT90132</td><td></td></tr><tr><td>Sodium citrate</td><td>Sigma</td><td>C7254</td><td></td></tr><tr><td>Ethanol 96%</td><td>Panreac</td><td>1,410,851,212</td><td></td></tr><tr><td>CFSE</td><td>Sigma</td><td>21888</td><td></td></tr><tr><td>RPMI</td><td>Lonza</td><td>BE12-115F</td><td></td></tr><tr><td>SDS</td><td>Bio-Rad</td><td>161-0418</td><td></td></tr><tr><td>Infinite M200</td><td>Tecan</td><td>M200</td><td>Multi mode microplate reader</td></tr><tr><td>Gel Doc 2000</td><td>Bio-Rad</td><td>2000</td><td>Gel documentation system</td></tr><tr><td>StepOnePlus</td><td>Applied Biosystems</td><td>StepOnePlus</td><td>qPCR system</td></tr><tr><td>MACSQuant Analyzer 10</td><td>Miltenyi Biotec</td><td>Analyzer 10</td><td>Cytometry equipment</td></tr><tr><td>ChemiDoc MP</td><td>Bio-Rad</td><td>MP</td><td>Chemiluminescence detection system</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PECAM-1</td><td>BD Biosciences</td><td>553370</td><td>Use at 10 &amp;micro;g/mL</td></tr><tr><td>ICAM-2</td><td>Biolegend</td><td>1054602</td><td>Use at 10 &amp;micro;g/mL</td></tr><tr><td>E-selectin</td><td>BD Biosciences</td><td>553749</td><td>Use at 10 &amp;micro;g/mL</td></tr><tr><td>VCAM-1</td><td>BD Biosciences</td><td>553330</td><td>Use at 10 &amp;micro;g/mL</td></tr><tr><td>ICAM-1</td><td>Becton Dickinson</td><td>553250</td><td>Use at 10 &amp;micro;g/mL</td></tr><tr><td>anti-rat IgG-FITC</td><td>Jackson Immuno Research</td><td>112-095-006</td><td>Use at 10 &amp;micro;g/mL</td></tr><tr><td>anti armenian hamster-FITC</td><td>Jackson Immuno Research</td><td>127-095-160</td><td>Use at 10 &amp;micro;g/mL</td></tr><tr><td>Rat IgG isotyope control</td><td>Invitrogen</td><td>10700</td><td>Use at 10 &amp;micro;g/mL</td></tr><tr><td>Armenian hamster IgG isotype control</td><td>Invitrogen</td><td>PA5-33220</td><td>Use at 10 &amp;micro;g/mL</td></tr><tr><td>P-I&amp;kappa;&amp;Beta;-&amp;alpha;</td><td>Cell Signaling</td><td>2859</td><td>Use at 10 &amp;micro;g/mL</td></tr><tr><td>&amp;beta;-Actin</td><td>Sigma</td><td>A5441</td><td>Use at 10 &amp;micro;g/mL</td></tr><tr><td>P-ERK</td><td>Cell Signaling</td><td>9101</td><td>Use at 10 &amp;micro;g/mL</td></tr><tr><td>anti-mouse HRP</td><td>GE Healthcare</td><td>LNXA931/AE</td><td>Use at 1:10,000</td></tr><tr><td>anti-rabbit HRP</td><td>GE Healthcare</td><td>LNA934V/AG</td><td>Use at 1:10,000</td></tr><tr><td>anti-rat HRP</td><td>Santa Cruz</td><td>Sc-3823</td><td>Use at 1:10,000</td></tr></tbody></table>

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.

Evaluation of LPS-induced endothelial cell activation by RT-qPCR
The serum starved MLEC-04 cells were stimulated by 100 ng/mL of LPS for 6 h, and the endothelial gene expression was assessed using RT-qPCR by comparing the expression of activation markers to the resting condition. As shown in Figure 1A, the LPS-incubated MLEC-04 cells induced the mRNA expression of selected adhesion molecules involved i...

Watch this Scientific Journal Video about Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response at JoVE.com

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

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 ...

screening-assays-to-characterize-novel-endothelial-regulators

Research

JoVE Journal

Immunology and Infection

6.5K Views.  Institute of Health Carlos III. Vasculaire endotheel bepaalt strak leukocyte werving. Onvoldoende leukocyte extravasation draagt bij tot de inflammatoire ziekten bij de mens. Daarom is zoeken naar nieuwe regelgevende elementen van endothelial activering om te ontwerpen van verbeterde therapieën voor inflammatoire aandoeningen. Hier beschrijven we een uitgebreide methodologie om te karakteriseren roman endothelial regelgevers die leukocyte handel tijdens ontsteking kunnen wijzigen.

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

Assessing Endothelial Vasodilator Function with the Endo-PAT 2000

The endothelium is a delicate monolayer of cells that lines all blood vessels, and which comprises the systemic and lymphatic capillaries. By virtue of the panoply of paracrine factors that it secretes, the endothelium regulates the contractile and proliferative state of the underlying vascular smooth muscle, as well as the interaction of the vessel wall with circulating blood elements. Because of its central role in mediating vessel tone and growth, its position as gateway to circulating immune cells, and its local regulation of hemostasis and coagulation, the the properly functioning endothelium is the key to cardiovascular health. Conversely, the earliest disorder in most vascular diseases is endothelial dysfunction.
In the arterial circulation, the healthy endothelium generally exerts a vasodilator influence on the vascular smooth muscle. There are a number of methods to assess endothelial vasodilator function. The Endo-PAT 2000 is a new device that is used to assess endothelial vasodilator function in a rapid and non-invasive fashion. Unlike the commonly used technique of duplex ultra-sonography to assess flow-mediated vasodilation, it is totally non-operator-dependent, and the equipment is an order of magnitude less expensive. The device records endothelium-mediated changes in the digital pulse waveform known as the PAT ( peripheral Arterial Tone) signal, measured with a pair of novel modified plethysmographic probes situated on the finger index of each hand. Endothelium-mediated changes in the PAT signal are elicited by creating a downstream hyperemic response. Hyperemia is induced by occluding blood flow through the brachial artery for 5 minutes using an inflatable cuff on one hand. The response to reactive hyperemia is calculated automatically by the system. A PAT ratio is created using the post and pre occlusion values. These values are normalized to measurements from the contra-lateral arm, which serves as control for non-endothelial dependent systemic effects. Most notably, this normalization controls for fluctuations in sympathetic nerve outflow that may induce changes in peripheral arterial tone that are superimposed on the hyperemic response.
In this video we demonstrate how to use the Endo-PAT 2000 to perform a clinically relevant assessment of endothelial vasodilator function.

A noninvasive procedure to assess endothelial function is demonstrated using the Endo-PAT 2000.

The endothelium is a delicate monolayer of cells that lines all blood vessels, and which comprises the systemic and lymphatic capillaries. By virtue of ...

Medicine

On-Chip Endothelial Inflammatory Phenotyping

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial dysfunction. However, early functional changes in endothelium that signify an individual's level of risk are not directly assessed clinically to help guide therapeutic strategy. Moreover, the regulation of inflammation by local hemodynamics contributes to the non-random spatial distribution of atherosclerosis, but the mechanisms are difficult to delineate in vivo. We describe a lab-on-a-chip based approach to quantitatively assay metabolic perturbation of inflammatory events in human endothelial cells (EC) and monocytes under precise flow conditions. Standard methods of soft lithography are used to microfabricate vascular mimetic microfluidic chambers (VMMC), which are bound directly to cultured EC monolayers.1 These devices have the advantage of using small volumes of reagents while providing a platform for directly imaging the inflammatory events at the membrane of EC exposed to a well-defined shear field. We have successfully applied these devices to investigate cytokine-,2 lipid-3, 4 and RAGE-induced5 inflammation in human aortic EC (HAEC). Here we document the use of the VMMC to assay monocytic cell (THP-1) rolling and arrest on HAEC monolayers that are conditioned under differential shear characteristics and activated by the inflammatory cytokine TNF-&alpha;. Studies such as these are providing mechanistic insight into atherosusceptibility under metabolic risk factors.

Microfluidic flow chambers etched by photolithography and fabricated from PDMS are applied to probe functional outcomes associated with EC dysfunction and inflammation. In a representative experiment, the ability of differential shear stress to modulate monocytic cell adhesion to cytokine activated EC monolayers is demonstrated.

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial ...

Bioengineering

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a &lt;1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes.
In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts.
In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage.

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and ...

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

Developmental Biology

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 ...

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells (hiPSC-ECs)

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected tissues via a well-characterized cascade of pro-adhesive receptors which are upregulated on the leukocyte cell surface upon the inflammatory trigger. Inflammatory responses differ between individuals in the population and the genetic background can contribute to these differences. Human induced pluripotent stem cells (hiPSCs) have been shown to be a reliable source of ECs (hiPSC-ECs), thus representing an unlimited source of cells that capture the genetic identity and any genetic variants or mutations of the donor. hiPSC-ECs can therefore be used for modeling inflammatory responses in donor-specific cells. Inflammatory responses can be modeled by determining leukocyte adhesion to the hiPSC-ECs under physiological flow. This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells hiPSC-ECs

This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected ...

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis1. Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor.

This manuscript describes a tube formation assay to quantify the effects of a given compound or condition on angiogenesis by using endothelial cell tube formation in a controlled environment.

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by ...

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 ...

Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood coagulation. This phenomenon is observed in many clinical situations, like organ transplantation in the presence of pre-formed anti-donor antibodies, including xenotransplantation, as well as in ischemia/reperfusion injury. In order to reduce animal experimentation according to the 3R standards (reduction, replacement and refinement), in vitro models to study the effect of endothelial cell activation on blood coagulation would be highly desirable. However, common flatbed systems of endothelial cell culture provide a surface-to-volume ratio of 1 - 5 cm2 of endothelium per mL of blood, which is not sufficient for natural, endothelial-mediated anticoagulation. Culturing endothelial cells on microcarrier beads may increase the surface-to-volume ratio to 40 - 160 cm2/mL. This increased ratio is sufficient to ensure the &#34;natural&#34; anticoagulation of whole blood, so that the use of anticoagulants can be avoided. Here an in vitro microcarrier-based system is described to study the effects of genetic modification of porcine endothelial cells on coagulation of whole, non-anticoagulated human blood. In the described assay, primary porcine aortic endothelial cells, either wild type (WT) or transgenic for human CD46 and thrombomodulin, were grown on microcarrier beads and then exposed to freshly drawn non-anticoagulated human blood. This model allows for the measurement and quantification of cytokine release as well as activation markers of complement and coagulation in the blood plasma. In addition, imaging of activated endothelial cell and deposition of immunoglobulins, complement- and coagulation proteins on the endothelialized beads were performed by confocal microscopy. This assay can also be used to test drugs which are supposed to prevent endothelial cell activation and, thus, coagulation. On top of its potential to reduce the number of animals used for such investigations, the described assay is easy to perform and consistently reproducible.

We present an in vitro model which allows the study and analysis of coagulation in whole, non-anticoagulated blood. Anticoagulation in the system depends on the natural anticoagulation effect of healthy endothelial cells and endothelial cell activation will result in clotting.

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood ...

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 ...

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

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