Funding for this project was provided by an American Cancer Society Research Scholar Award, (06-024-01-CSM to Charles J. Dimitroff); a National Institutes of Health/National Cancer Institute grant (RO1CA118124 to Charles J. Dimitroff); and a National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases grant (P30 AR042689 to Dr. Thomas S. Kupper; Brigham and Women&rsquo;s Hospital, Boston, MA).

1. Preparation of hBMEC (HBMEC-60 Cell) Monolayer on Petri Dishes for Flow Chamber <ol> <li>Coat 35 x 10mm tissue-culture dishes with 2ml of 20&micro;g/ml fibronectin (in PBS) overnight at 4&deg;C or for 3 hours at 37&deg;C.</li> <li>Aspirate fibronectin solution from dishes and add 1.5 x105 HBMEC-60 cells [Medium 199 with HEPES &amp; Glutamine,10% FBS,10% Human Serum, 5 units/ml heparin, 1ng/ml recombinant human fibroblast growth factor,1%Penicillin-Streptomycin] to the dish and allow them to grow to &gt;90% confluence. (This takes 2 days)</li> <li>Aspirate growth media and, to up-regulate E-selectin expression, add fresh medium with 50ng/mL IL-1&beta; for 4-6 hrs. Cell monolayers are now ready for rolling assay. To block E-selectin function, neutralizing anti-human E-selectin moAb (clone BBIG-E4(5D11)) can be added at 20&micro;g/ml for ~1hr at 37&deg;C.</li> </ol> 2. Preparation of Human Hematopoietic Progenitor KG1a Cells for Flow Chamber <ol> <li>Human hematopoietic progenitor KG1a cells are grown to confluency (1x106 cells/ml) in RPMI-1640 with glutamine and 10% FBS/1% penicillin-streptomycin and are pipetted directly from the flask for use in flow chamber assay.</li> <li>Cell number is calculated using a hemacytometer and then cells are re- suspended at 1 x 106 cells/mL in HBSS with 10mM HEPES (H/H Buffer) and 2mM CaCl2</li> <li>Cells are stored on ice until use in assay.</li> </ol> 3. Preparation of Microscope, Parallel-Plate Flow Chamber and Syringe Pump (Figure 1) <img src="/files/ftp_upload/old/1009_2009_1_26_7/1009_figure1.png" alt="Figure 1" width="600" height="409" /> Figure 1.Illustration of Parallel-Plate Flow Chamber Analysis. Inverted microscope, Harvard Syringe Pump, computer/camera and parallel-plate flow chamber are placed in an optimal arrangement for efficient and reproducible cell analysis. <ol> <li>Turn on power and light source to inverted microscope and power to Syringe Pump and select the 10X Objective on the microscope.</li> <li>Place 50mL conical tube in holder and fill with H/H and 2mM CaCl2.</li> <li>Place 5ml plastic test tubes in test tube holder below the 50mL conical tube</li> <li>Place 60mL syringe in Syringe Pump (Be sure that both the plunger and body of syringe are both secured).</li> <li>Attach 3-way stop-cock to end of 60mL syringe and attach 5mL syringe to 3-way stop-cock.</li> <li>Place Parallel Flow Chamber apparatus (GlycoTech, Inc.) on microscope stage with input tube to the right, output tube to the left and vacuum tube to the back.</li> <li>Attach vacuum tubing to vacuum and open air valve to allow parallel plate apparatus to adhere to the stage. (This tests if the vacuum skirt on the flow chamber is air-tight.) Turn off air valve.</li> <li>Attach output tubing line (on left of microscope) to 3-way stop-cock. Apparatus is now prepared for placing on HBMEC-60 monolayer plate.</li> <li>Place HBMEC-60 monolayer plate and place on center of microscope.</li> <li>Turn on vacuum and position the flow chamber apparatus above the plate such that the monolayer plate is in the center.</li> <li>Gently lower the flow chamber apparatus onto the HBMEC-60 monolayer plate and allow flow chamber apparatus to suction down. (There should be no sounds of air escaping and at this point you may need to apply pressure to chamber to compress the rubber gasket.)</li> <li>Once vacuum has been established, place input tube (on the right) into the 50mL conical tube containing H/H with 2mM CaCl2.</li> <li>Open 3-way stop-cock to allow flow into the 60mL syringe and place pump in "pump mode."</li> <li>To remove air from input/output lines without lifting the cells on the plate, use the Syringe Pump set at 2mL/minute to fill the intake tube, then quickly set to 0.5 mL/minute just before the fluid reaches the apparatus. It is imperative to prime this set up carefully and avoid air bubbles which will lift the monolayer from the plate.</li> <li>Once fluid is seen to move into the 60ml syringe, the flow chamber and Syringe Pump have been primed and the Flow chamber is ready for use. This set up will be repeated for each cell input run and for each new plate needed.</li> </ol> 4. Capturing Leukocyte Rolling Events using NIS Elements <ol> <li>Open NIS Elements on desktop.</li> <li>Press "play" icon for live image. Auto exposure will allow you to see the plate easier for now and is important for focusing.</li> <li>Be sure that microscope is focused properly on the cell monolayer.</li> <li>Once focused, re-set exposure to 1 frame/ second and adjust light on the microscope. The image should appear on the computer and no longer be visible in the microscope viewfinder due to the low light level. Light histogram can then be adjusted to remove background exposure or improve cell clarity.</li> <li>Click on 2X2 bin or Live Bin to obtain picture that is ideal for movie capture</li> <li>Click on "Macro" on menu bar and select "write to port" ; select port "com3," and "open". (This opens the port to coordinate with the Syringe Pump.</li> <li>Pipette 1mL of KG1a cell suspension into the 5ml test tube near the input tubing line and then place input tubing line to the bottom of the test tube.</li> <li>Go to menu bar, click on "Capture" ; "time-lapse acquisition."</li> <li>A new menu will pop up. Select the protocol lasting 210 seconds, this is the length of the program for rolling on HBMEC-60 Cells. (Each phase is programmed to take a picture every second for the duration of the pump protocol. It is also set up to send a command through the com3 port to start the pump.) If time-lapse is interrupted, the pump will not stop on its own and must be reset. Press Cancel to return to live image.</li> <li>Set the Syringe Pump to "Program Mode" according to instruction manual. (Program mode allows for manual programming of specific flow rates (i.e. shear stress levels) to be imparted within the chamber and dictate KG1a cell interactions over the HBMEC-60 cell monolayer.) Settings for KG1a cell rolling on hBMEC Cells were as follows 4.2 dynes/cm2 for 45 s, 0.3 dynes/cm2 for 60 s, and stepwise increases every 15 s to a maximum of 4.2 dynes/cm2</li> <li>Wall shear stress (Wst) in the chamber was calculated according to the following equation: Wst = 6&micro;Q/a2b, where &mu; is the estimated viscosity of the media (0.0076 P), Q is the volumetric flow rate in ml/s, a is the channel height (gasket thickness) in cm (0.03 cm), and b is the channel width in cm (0.5 cm). Flow rates were regulated using the program mode of the Syringe Pump.</li> <li>Microscope and computer are now ready to capture time-lapse photography. Press "start run" on the computer to begin acquiring data. While running the program, be sure to add media to the test tube containing KG1a cells in order to keep the input/output tubing full of medium (Make sure there are no air bubbles).</li> </ol> 5. Dependence of Terminal Sialylation and Surface Glycoprotein for E-selectin-Mediated KG1a Cell 5. Rolling (Figure 2; Videos Clips of 5.1-5.5) <ol> <li>Assaying of KG1a cell rolling on non-stimulated HBMEC-60 cells. (Negative control) KG1a cells were prepared and infused into the flow chamber over live non-IL-1&beta;-stimulated HBMEC-60 as described above.</li> <li>Assaying of KG1a cell rolling on HBMEC-60 cells pre-treated with IL-1&beta;. (E-selectin-dependence control) KG1a cells were prepared and infused into the flow chamber over live IL-1&beta;-stimulated HBMEC-60 as described above.</li> <li>Assaying of KG1a cell rolling on HBMEC-60 cells pre-treated with IL-1&beta; and then neutralizing anti-human E-selectin moAb. (E-selectin-dependence control) KG1a cells were prepared and infused into the flow chamber over live IL-1&beta;- stimulated HBMEC-60 pretreated with neutralizing anti-human E-selectin moAb as described above.</li> <li>Assaying of sialidase-digested KG1a cell rolling on IL-1&beta;-stimulated HBMEC-60. KG1a cells pretreated with 0.1U/ml Vibrio Cholerae neuraminidase for 1hr at 37&deg;C were prepared and infused into the flow chamber over live IL-1&beta;-stimulated HBMEC- 60 as described above.</li> <li>Assaying of protease-digested KG1a cell rolling on IL-1&beta;-stimulated HBMEC-60. KG1a cells pretreated with protease, 0.2U/ml Bromelain for 1hr at 37&deg;C, were prepared and infused into the flow chamber over live IL-1&beta;-stimulated HBMEC-60 as described above.</li> </ol> 6. Analysis of NIS-Elements-Captured KG1a Cells and Graphing of KG1a Cell Rolling Data <ol> <li>After time sequence is acquired, save the data.</li> <li>Go to "measure" tab on menu bar and select "define threshold." Move bars so that desired cells are colored in red. Select "all images" then "Okay". The entire time-lapse sequence should now be modified with visible red rolling cells.</li> <li>Go to "Measure" and select "restrictions." (Restrictions allow the user to exclude thresholded objects that do not represent rolling cells. This is primarily background created by the HBMEC-60 monolayer. ) Select "area" and input min and max values for the area for rolling cells. Repeat this step for "elongation" and "circularity."</li> <li>Select "measure" and select "create binary using restrictions", and set the restriction values to optimize NIS-Elements software recognition of rolling cells on monolayer of HBMEC-60 cells.</li> <li>Go back to "measure", select "Field data", and then select "reset data". Close this menu.</li> <li>Return to "Measure" and select "scan field".</li> <li>Open "Measure"; "Field data", select "number or Objects" and then select "all fields" and export data to Microsoft Excel.</li> <li>Select "clear data" to prepare NIS-Elements cell acquisition for next data collection. </li> </ol> Results: <img src="/files/ftp_upload/old/1009_2009_1_26_8/1009_figure3.png" alt="Figure 3" width="600" height="382" /> Figure 3. Parallel-Plate Flow Chamber Analysis of E-selectin-Binding Activity on KG1a Cells. KG1a cells treated with sialidase or protease were analyzed for rolling efficiency on monolayers of HBMEC-60 cells pre-treated with IL-1&beta;. Negative control rolling experiments were also conducted by analyzing KG1a cell rolling on non-IL-1&beta;-stimulated HBMEC -60 cells or on IL-1&beta;-stimulated HBMEC-60 cells treated with neutralizing anti-human E-selectin moAb (5D11 ). Cell rolling frequencies were performed in triplicate and analyzed with NIS-Elements software. (* Statistical significance; p&lt;0.001) &nbsp; In this video-based report, we provided a visual depiction of how to analyze leukocyte &ndash; endothelial cell interactions under physiologic shear stress conditions. In particular, we analyzed the role of E-selectin &ndash; E-selectin ligands in mediating leukocyte tethering and rolling, an adhesive behavior necessary for commitment of secondary more stable binding activities through receptors, such as integrins and hyaluronan receptors. Using the parallel-plate flow chamber adapted for use with an inverted microscope, Harvard Syringe Pump, video camera and computer/cell acquisition hardware, we conducted experiments wherein E-selectin ligand+ KG1a cells were used as our leukocyte model and hBMEC cells (HBMEC-60) stimulated with pro-inflammatory cytokine, IL-1&beta;, were used as our E-selectin+ human endothelial cell model (1-6). As previously reported, we observed robust KG1a cell rolling on IL-1&beta;-stimulated HBMEC-60 cells over a range of shear stress levels in an E-selectin-dependent manner (Figure 3)(Statistically significant difference when compared with KG1a cell rolling on non-IL-1&beta; stimulated HBMEC-60 cells). Negative controls, which consisted of KG1a cell rolling on non-IL-1&beta;-stimulated HBMEC-60 cells or on IL-1&beta;-stimulated HBEMC-60 cells pretreated with anti-human E-selectin mAb, were performed in parallel to demonstrate the dependence of cytokine stimulation and E-selectin expression for rolling activity (Figure 3). Furthermore, rolling analysis of KG1a cells pretreated with Vibrio cholerae neuraminidase (sialidase) or with bromelain, a non-specific protease known for digesting surface E-selectin glycoprotein ligand, highlighted the importance of terminal sialic acid residues and surface glycoprotein for E-selectin ligand activity (2,3).

<ol>
	<li>Dimitroff, C. J., Kupper, T. S., Sackstein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+leukocytic+migration+to+inflamed+skin+with+a+novel+fluorosugar+modifier+of+cutaneous+lymphocyte-associated+antigen.">Prevention of leukocytic migration to inflamed skin with a novel fluorosugar modifier of cutaneous lymphocyte-associated antigen.</a> J. Clin. Invest. 112, 1008-1018 .</li><li>Dimitroff, C. J., Lechpammer, M., Long-Woodward, D., Kutok, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rolling+of+human+bone-metastatic+prostate+tumor+cells+on+human+bone+marrow+endothelium+under+shear+flow+is+mediated+by+E-selectin.">Rolling of human bone-metastatic prostate tumor cells on human bone marrow endothelium under shear flow is mediated by E-selectin.</a> Cancer Res. 64, 5261-5269 (2004).</li><li>Dimitroff, C. J., Descheny, L., Trujillo, N., Kim, R., Nguyen, V., Huang, W., Pienta, K. J., Kutok, J. L., Rubin, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+leukocyte+E-selectin+ligands,+P-selectin+glycoprotein+ligand-1+and+E-selectin+ligand-1,+on+human+metastatic+prostate+tumor+cells.">Identification of leukocyte E-selectin ligands, P-selectin glycoprotein ligand-1 and E-selectin ligand-1, on human metastatic prostate tumor cells.</a> Cancer Res. 65, 5750-5760 (2005).</li><li>Descheny, L., Gainers, M. E., Walcheck, B., Dimitroff, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ameliorating+Skin-Homing+Receptors+on+Malignant+T+cells+with+a+Fluorosugar+Analog+of+N-acetylglucosamine:+P-selectin+Ligand+is+a+More+Sensitive+Target+than+E-selectin+Ligand.">Ameliorating Skin-Homing Receptors on Malignant T cells with a Fluorosugar Analog of N-acetylglucosamine: P-selectin Ligand is a More Sensitive Target than E-selectin Ligand.</a> J Invest Dermatol. 126 (9), 2065-2073 (2006).</li><li>Gainers, M. E., Descheny, L., Barthel, S. B., Liu, L., Wurbel, M., Dimitroff, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin-homing+receptors+on+effector+leukocytes+are+differentially+sensitive+to+glyco-metabolic+antagonism+in+allergic+contact+dermatitis.">Skin-homing receptors on effector leukocytes are differentially sensitive to glyco-metabolic antagonism in allergic contact dermatitis.</a> J. Immunology. 179 (12), 8509-8518 (2007).</li><li>Rood, P. M., Calafat, J., von dem Borne, A. E., Gerritsen, W. R., van der Schoot, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+of+human+bone+marrow+endothelial+cells:+characterisation+of+new+cell+lines.">Immortalization of human bone marrow endothelial cells: characterisation of new cell lines.</a> Eur J Clin Invest. 30 (7), 618-629 (2000).</li></ol>

The authors have no conflicts of interest or disclosures.

Parallel-plate flow chamber analysis is a specialized in vitro assay system used for studying leukocyte &ndash; endothelial adhesive interactions under shear stress conditions similar those imparted on the surface of a post-capillary venule. As depicted here in our visual presentation, we demonstrate the value of this system for investigating the role of E-selectin &ndash; E-selectin ligands in mediating leukocyte tethering and rolling on the surface of activated microvascular endothelium. We also reveal the application of protease, glycosidase and blocking antibody treatments for elucidating the role of E-selectin and its ligands in this system. These analyses are highly reproducible and help form an experimental basis for studying E-selectin &ndash; E-selectin ligand function in vivo.
In addition to studying cell adhesion with monolayers of cytokine-activated microvascular endothelial cells (hBMEC or human umbilical vein or dermal microvascular endothelial cells), assays can be performed using purified recombinant human E-selectin or E-selectin-Ig chimeric molecules as a substrate for cell adhesion. Other adhesive interactions mediated through leukocyte (L-)-selectin and platelet (P)-selectin can also be assayed using parallel-plate flow chamber technology. By varying the type of cell monolayer/protein substrate or of input leukocyte/selectin-transfectant cell models, investigators can study the role of these interactions under physiologic shear stress conditions.

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		<div>
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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>SIGMA</td>
<td>F6178-500ML</td>
<td>&nbsp;</td>
<tr>
<td>DPBS</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>14190</td>
<td>&nbsp;</td>
<tr>
<td>0.5 M EDTA</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15575-038</td>
<td>&nbsp;</td>
<tr>
<td>1M HEPES</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15630</td>
<td>&nbsp;</td>
<tr>
<td>Parallel-Plate Flow Chamber Kit</td>
<td>&nbsp;</td>
<td>GlycoTech</td>
<td>31-001</td>
<td>&nbsp;</td>
<tr>
<td>PHD2000 Programmable Syringe Pump</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>PHD 2000</td>
<td>&nbsp;</td>
<tr>
<td>35x10mm Tissue Culture Dish</td>
<td>&nbsp;</td>
<td>BD FALCON</td>
<td>353001</td>
<td>&nbsp;</td>
<tr>
<td>Fibronectin , from human plasma</td>
<td>&nbsp;</td>
<td>SIGMA</td>
<td>86088-83-7 F2006-2MG</td>
<td>&nbsp;</td>
<tr>
<td>Interleukin-1_, Human; Recombinant</td>
<td>&nbsp;</td>
<td>SIGMA</td>
<td>I9401-5UG</td>
<td>&nbsp;</td>
<tr>
<td>HBSS (Hank s Buffered Saline Solution)</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>14170</td>
<td>&nbsp;</td>
<tr>
<td>Medium 199 w/ HEPES & Glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12320-039</td>
<td>&nbsp;</td>
<tr>
<td>Human Serum</td>
<td>&nbsp;</td>
<td>Innovative Research, Inc.</td>
<td>IPLA-SER1</td>
<td>&nbsp;</td>
<tr>
<td>Heparin</td>
<td>&nbsp;</td>
<td>Abraxis Pharmaceutical Products</td>
<td>504011</td>
<td>&nbsp;</td>
<tr>
<td>Recombinant human fibroblast growth factor</td>
<td>&nbsp;</td>
<td>R&D Systems</td>
<td>233-FB</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin Streptomycin</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Plastic Test tubes</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>352008</td>
<td>&nbsp;</td>
<tr>
<td>Anti-human E-selectin clone BBIG-E4(5D11)</td>
<td>&nbsp;</td>
<td>R&D Systems	</td>
<td>BBA16</td>
<td>&nbsp;</td>
<tr>
<td>Bromelain</td>
<td>&nbsp;</td>
<td>SIGMA</td>
<td>B-4882</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
				 	
	

		</div>

analysis of physiologic e-selectin-mediated leukocyte rolling on microvascular endothelium

E-selectin is a type-1 membrane protein on microvascular endothelial cells that helps initiate recruitment of circulating leukocytes to cutaneous, bone and inflamed tissues.  E-selectin expression is constitutive on dermal and bone microvessels and is inducible by pro-inflammatory cytokines, such as IL-1&#945;/  and TNF-&#945;, on microvessels in inflamed tissues.  This lectin receptor mediates weak binding interactions with carbohydrate counter-receptor ligands on circulating leukocytes, which results in a characteristic  rolling  behavior.  Because these interactions precede more stable adhesive events and diapedesis activity, characterization of leukocyte rolling activity and identification of leukocyte E-selectin ligands have been major goals in studies of leukocyte trafficking and inflammation and in the development of anti-inflammatory therapeutics (1-5).  The intent of this report is to provide a visual, comprehensive description of the most widely-used technology for studying E-selectin   E-selectin ligand interactions under physiologic blood flow conditions.  Our laboratory in conjunction with the Harvard Skin Disease Research Center uses a state-of-the-art parallel-plate flow chamber apparatus accompanied by digital visualization and new recording software, NIS-Elements.  This technology allows us to analyze adhesion events in real time for onscreen visualization as well as record rolling activity in a video format.  Cell adhesion parameters, such as rolling frequency, shear resistance and binding/tethering efficiency, are calculated with NIS-Elements software, exported to an Excel spreadsheet and subjected to statistical analysis.  In the demonstration presented here, we employed the parallel-plate flow chamber to investigate E-selectin-dependent leukocyte rolling activity on live human bone marrow endothelial cells (hBMEC).  Human hematopoietic progenitor KG1a cells, which express a high level of E-selectin ligand, were used as our leukocyte model, while an immortalized hBMEC cell line, HBMEC-60 cells, was used as our endothelial cell model (6).  To induce and simulate native E-selectin expression in the flow chamber, HBMEC-60 cells were first activated with IL-1 .  Our video presentation showed that parallel-plate flow analysis is a suitable method for studying physiologic E-selectin-mediated leukocyte rolling activities and that functional characterization of leukocyte E-selectin ligand(s) in the flow chamber can be ascertained by implementing protease or glycosidase digestions.

Watch this Scientific Journal Video about Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium at JoVE.com

Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium

This report provides a visual depiction of parallel-plate flow chamber analysis for studying leukocyte endothelial interactions under physiologic shear stress. This method is particularly useful for investigating the role of endothelial (E)-selectin and leukocyte E-selectin ligands that trigger leukocyte rolling on endothelial cell surfaces.

E-selectin is a type-1 membrane protein on microvascular endothelial cells that helps initiate recruitment of circulating leukocytes to cutaneous, bone ...

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19.0K Views.  Brigham and Women's Hospital. This report provides a visual depiction of parallel-plate flow chamber analysis for studying leukocyte endothelial interactions under physiologic shear stress. This method is particularly useful for investigating the role of endothelial (E)-selectin and leukocyte E-selectin ligands that trigger leukocyte rolling on endothelial cell surfaces.

Video: Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g. drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptome analysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism1,2 . RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptome analysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence3.
Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled "RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin,"4 in which we successfully performed the first complete transcriptome analysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases.
The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state.

This protocol presents a complete and detailed procedure to apply RNA-seq, a powerful next-generation DNA sequencing technology, to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is generalizable to various cells or tissues affected by different reagents or disease states.

The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the ...

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.