This work was supported by CIHR MOP 142307 to P.A.M. H.S.J.M. is a recipient of a Canadian Institutes of Health Research Training Program in Regenerative Medicine Fellowship. H.S.J.M., A.N.S., K.H.K., and M.K.D. are recipients of the Queen Elizabeth II Graduate Scholarships in the Science and Technology.

1. Preparation of Exogenous Reference RNA
NOTE: Choose an exogenous reference RNA that does not exist in the species or model of interest. For mammalian systems, firefly luciferase RNA may be used.
<ol>
	<li>Linearization of exogenous reference RNA plasmid
	<ol>
		<li>Prepare exogenous reference RNA at least 48 h prior to the anticipated RNA extraction. Obtain or manufacture a cDNA clone of the chosen exogenous reference RNA, such as a firefly luciferase cDNA clone in a plas...

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenic+patterning+by+STEEL,+an+endothelial-enriched+long+noncoding+RNA.">Angiogenic patterning by STEEL, an endothelial-enriched long noncoding RNA.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (10), 2401-2406 (2018).</li><li>Won, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relative+reduction+of+endothelial+nitric-oxide+synthase+expression+and+transcription+in+atherosclerosis-prone+regions+of+the+mouse+aorta+and+in+an+in+vitro+model+of+disturbed+flow.">Relative reduction of endothelial nitric-oxide synthase expression and transcription in atherosclerosis-prone regions of the mouse aorta and in an in vitro model of disturbed flow.</a> The American Journal of Pathology. 171 (5), 1691-1704 (2007).</li><li>Baeyens, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+remodeling+is+governed+by+a+VEGFR3-dependent+fluid+shear+stress+set+point.">Vascular remodeling is governed by a VEGFR3-dependent fluid shear stress set point.</a> eLIFE. 4, 04645 (2015).</li><li>Davies, P. F., Civelek, M., Fang, Y., Fleming, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+atherosusceptible+endothelium:+endothelial+phenotypes+in+complex+haemodynamic+shear+stress+regions+in+vivo.">The atherosusceptible endothelium: endothelial phenotypes in complex haemodynamic shear stress regions in vivo.</a> Cardiovascular Research. 99 (2), 315-327 (2013).</li><li>Lane, W. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel-plate+flow+chamber+and+continuous+flow+circuit+to+evaluate+endothelial+progenitor+cells+under+laminar+flow+shear+stress.">Parallel-plate flow chamber and continuous flow circuit to evaluate endothelial progenitor cells under laminar flow shear stress.</a> Journal of Visualized Experiments. (59), 3349 (2012).</li><li>Gray, K. M., Stroka, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+endothelial+cell+mechanosensing:+New+insights+gained+from+biomimetic+microfluidic+models.">Vascular endothelial cell mechanosensing: New insights gained from biomimetic microfluidic models.</a> Seminars in Cell and Developmental Biology. 71, 106-117 (2017).</li><li>Bussolari, S. R., Dewey, C. F., Gimbrone, 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=Apparatus+for+subjecting+living+cells+to+fluid+shear+stress.">Apparatus for subjecting living cells to fluid shear stress.</a> Review of Scientific Instruments. 53 (12), 1851-1854 (1982).</li><li>Resnick, N., Gimbrone, 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=Hemodynamic+forces+are+complex+regulators+of+endothelial+gene+expression.">Hemodynamic forces are complex regulators of endothelial gene expression.</a> The FASEB Journal. 9 (10), 874-882 (1995).</li><li>Malek, A. M., Alper, S. L., Izumo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+shear+stress+and+its+role+in+atherosclerosis.">Hemodynamic shear stress and its role in atherosclerosis.</a> The Journal of the American Medical Association. 282 (21), 2035-2042 (1999).</li><li>DeStefano, J. G., Xu, Z. S., Williams, A. J., Yimam, N., Searson, P. 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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+to+overcome+the+inhibitory+effect+of+heparin+on+DNA+amplification.">A simple method to overcome the inhibitory effect of heparin on DNA amplification.</a> Cellular Oncology (Dordrecht). 38 (6), 493-495 (2015).</li><li>Collins, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haemodynamic+and+extracellular+matrix+cues+regulate+the+mechanical+phenotype+and+stiffness+of+aortic+endothelial+cells.">Haemodynamic and extracellular matrix cues regulate the mechanical phenotype and stiffness of aortic endothelial cells.</a> Nature Communications. 5, 3984 (2014).</li><li>Fichtlscherer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+microRNAs+in+patients+with+coronary+artery+disease.">Circulating microRNAs in patients with coronary artery disease.</a> Circulation Research. 107 (5), 677-684 (2010).</li><li>Smith, R. 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The authors have nothing to disclose.

Shear stress is a physiologic condition that modulates endothelial function, in part, by affecting steady-state gene expression2,5. Models of gene regulation in various shear stress conditions will contribute to a greater understanding of endothelial function. This pragmatic workflow includes a flow circuit using a parallel-plate flow chamber adapted from Lane et al.9 and represents laminar, non-pulsatile flow. The overall set-up ...

Vascular endothelial cells form the inner cellular lining of blood vessels in the closed cardiovascular system of higher species. They form the interface between the blood and tissues and are characterized by luminal and abluminal surfaces. The endothelium is a diverse, active, and adaptive system that regulates blood flow, nutrient trafficking, immunity, and the growth of new blood vessels1. In the body, endothelial cells normally exist in an environment where they are exposed to the frictional force of circulation, shear stress2. Shear stress is an important regulator of endothelial cell gene expression3, and endothelial cells attempt to maintain shear stress within a given range2,4. Endothelial cells demonstrate angiogenic patterning in the absence of shear stress5 that can improve tissue perfusion. Regional patterns of disturbed flow and altered shear stress are associated with the expression of inflammatory genes6 and the development of atherosclerosis7,8. Thus, models that include shear stress are a major component of understanding endothelial gene regulation.
We describe a method for studying the gene regulation in vascular endothelial cells under shear stress. This system uses non-pulsatile flow and mimics fluid shear stress levels and oxygen concentration that model conditions for arterial endothelial cells. This protocol includes details of methods for the gene knockdown using RNA interference (RNAi), the set-up for the application of shear stress using the parallel-plate flow apparatus, and methods for the spike-in of an exogenous reference RNA prior to analysis by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR). This pipeline is used for studying gene regulation in endothelial cells in the presence and absence of laminar shear stress and includes an adaptation of the parallel-plate flow apparatus described by Lane et al.9. This particular set-up was designed to facilitate the simultaneous assessment of multiple experimental conditions that allows direct comparison of shear stress conditions, as well as the normalization of RNA analysis. A large heated unit with controlled humidity is utilized to allow multiple separate flow chambers and pumps to be running simultaneously with flow rates monitored for each flow chamber assembly in real-time. The application of this set-up is used for gene knockdown using RNAi in the setting of laminar flow/shear stress, but aspects of this protocol can be applied to any assessment of RNA expression.
Common approaches to the application of shear stress for endothelial cells include microfluidic systems10, a cone-and-plate viscometer11, and a parallel-plate flow chamber12. Microfluidic systems from various manufacturers have been useful in studying mechanobiology and mechanotransduction in multiple cell and tissue types and a variety of biophysical stimuli. For endothelial cells, they have been used to study endothelial cells in isolation, as well as the interaction of endothelial cells and the trafficking of immune or tumor cells10. However, these systems are less suitable for the recovery of large numbers of cells9. Both the cone-and-plate viscometer and parallel-plate flow chambers allow the recovery of large numbers of cells in confluent monolayers12. These systems can generate a range of shear forces and patterns12. The parallel-plate flow chamber assembly9 has the advantage that real-time imaging can be performed through the glass window to evaluate cellular morphology at any time point. Furthermore, the perfusate can be collected under sterile conditions. For the system presented here, the flow can also be monitored in real-time and in a multi-chamber set-up, which facilitates the maintenance of shear conditions between chambers.
For representative experiments, human umbilical vein endothelial cells (HUVEC), which represent a macrovascular endothelial cell type, are used, and the shear stress conditions we use (1 Pa) reflect arterial conditions (0.1 - 0.7 Pa). However, this protocol can be used with other endothelial cell types, and the shear stress conditions can be adjusted according to the experimental question. For example, the evaluation of human endothelial cells in conditions that model venous circulation would require lower levels of shear stress (1 - 6 Pa) and studies that model microvascular circulation have utilized shear stress levels of 0.4 - 1.2 Pa13,14. In addition, shear stress can vary even between endothelial cells within the same blood vessel6. In the current set-up, a single monitoring system is used that can simultaneously monitor four separate flow loops. For labs that need more flow loops, there is space in the dedicated environment for an additional monitoring system.
RT-qPCR is used for the absolute quantitation of gene expression in the setting of shear stress. The relative expression of target genes is often used to compare RNA expression across conditions. Some RNA species can exist at very low quantities or be absent, thus complicating relative measurements. For example, long noncoding RNAs in endothelial cells can exert potent effects at relatively low copy numbers per cell5. In addition, differences in primer efficiency can lead to an inaccurate interpretation from utilizing the delta-delta cycle threshold (Ct) method to analyze the data. To address this concern, we perform absolute quantitation by generating a standard curve using a known quantity of plasmid DNA. Furthermore, complementary DNA (cDNA) synthesis is an inefficient process, and differences in cDNA efficiency can account for differences in RNA expression between conditions and between samples15. The application of shear stress and/or transfection reagents can affect cell proliferation, apoptosis, and viability, or add components that may interfere with RNA isolation and/or cDNA synthesis. To account for the possibility of bias from RNA isolation and cDNA synthesis, we use a spike-in RNA control synthesized in the lab, added at the time of RNA extraction and measured with each cDNA synthesis via RT-qPCR. This allows not only the adjustment for technical differences in RNA extraction and cDNA synthesis but also allows the calculation of absolute quantities per cell, when the cell count is known.
This system uses additional steps to maintain similarity or account for technical differences between conditions. We particularly emphasize these steps because of the complex nature of these experiments, which involve multiple physical set-ups and experimental conditions that can lead to experimental variability.

<table>
	<tbody>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>gibco</td>
			<td>25300-062</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Syringe</td>
			<td>BD</td>
			<td>302995</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mm2 Culture Dish</td>
			<td>Sarstedt</td>
			<td>83.3902</td>
			<td></td>
		</tr>
		<tr>
			<td>30 mL Syringe</td>
			<td>BD</td>
			<td>302832</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Way Stopcocks</td>
			<td>Discofix</td>
			<td>D500</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BEACH</td>
			<td>Darwin Chambers Company</td>
			<td>MN: HO85, SN: 4947549</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Scrapers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Meter</td>
			<td>BioSphenix, Ltd.</td>
			<td>MN: P120, SN: 0342</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Sensor</td>
			<td>BioSphenix, Ltd.</td>
			<td>MN: C700, SN: 52852</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td>gibco</td>
			<td>15230-170</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline (DPBS) -/-</td>
			<td>gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium 2</td>
			<td>Promo Cell</td>
			<td>C-22011</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium 2 Supplement Mix</td>
			<td>Promo Cell</td>
			<td>C-39216</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin (pure)</td>
			<td>Sigma-Aldrich</td>
			<td>11051407001</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter (0.20 um)</td>
			<td>Sarstedt</td>
			<td>83.1826.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Dampener and Cap</td>
			<td>U of T glass blowing shop</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Meter: 400 Series Console</td>
			<td>Transonic Scisense Inc.</td>
			<td>T402</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Meter: 400 Series Tubing</td>
			<td>Transonic Scisense Inc.</td>
			<td>TS410</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Reservoir and Cap</td>
			<td>U of T glass blowing shop</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Sensor</td>
			<td>Transonic Scisense Inc.</td>
			<td>ME4PXL</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotemp 737F Oven</td>
			<td>Fisher Scientific</td>
			<td>FI-737F</td>
			<td></td>
		</tr>
		<tr>
			<td>J cloth</td>
			<td>J cloth</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slide (25 x 75 x 1 mm)</td>
			<td>Fisherfinest</td>
			<td>12-544-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Paper sterilization pouch</td>
			<td>Cardinal Health</td>
			<td>92713</td>
			<td></td>
		</tr>
		<tr>
			<td>Pump (Masterflex L/S Economy Drive)</td>
			<td>Cole-Parmer</td>
			<td>7554-90</td>
			<td></td>
		</tr>
		<tr>
			<td>Pump Head (Masterflex L/S Easy Load)</td>
			<td>Cole-Parmer</td>
			<td>7518-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Rectangular 4 Well Dish</td>
			<td>Thermo Scientific</td>
			<td>267061</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Tubing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex C-Flex L/S 25 Soft Tubing</td>
			<td>Cole-Parmer</td>
			<td>06424-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex C-Flex L/S 14 Soft Tubing</td>
			<td>Cole-Parmer</td>
			<td>06424-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex C-Flex L/S 16 Soft Tubing</td>
			<td>Cole-Parmer</td>
			<td>06424-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex PharMed BPT L/S 13 Hard Tubing</td>
			<td>Cole-Parmer</td>
			<td>06508-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex PharMed BPT L/S 14 Hard Tubing</td>
			<td>Cole-Parmer</td>
			<td>06508-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Luer </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3/16&#34; Male Luer</td>
			<td>Cole-Parmer</td>
			<td>45518-08</td>
			<td>For #25 tubing</td>
		</tr>
		<tr>
			<td>1/8&#34; Male Luer</td>
			<td>Cole-Parmer</td>
			<td>30800-24</td>
			<td>For #16 tubing</td>
		</tr>
		<tr>
			<td>1/8&#34; Female Luer</td>
			<td>Cole-Parmer</td>
			<td>30800-08</td>
			<td>For #16 tubing</td>
		</tr>
		<tr>
			<td>1/16&#34; Male Luer</td>
			<td>Cole-Parmer</td>
			<td>45518-00</td>
			<td>For #14 tubing</td>
		</tr>
		<tr>
			<td>1/16&#34; Female Luer</td>
			<td>Cole-Parmer</td>
			<td>45508-00</td>
			<td>For #14 tubing</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Knockdown reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oligofectamine Reagent</td>
			<td>Invitrogen</td>
			<td>12252-011</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium</td>
			<td>gibco</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>In vitro transcription </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Generuler 1kb+ DNA ladder</td>
			<td>Thermo Scientific</td>
			<td>SM1331</td>
			<td></td>
		</tr>
		<tr>
			<td>MEGAclear Kit</td>
			<td>Ambion</td>
			<td>AM1908</td>
			<td></td>
		</tr>
		<tr>
			<td>mMESSSEGE mMACHINE SP6 Transcription Kit</td>
			<td>Ambion</td>
			<td>AM1340</td>
			<td></td>
		</tr>
		<tr>
			<td>pSP-luc+</td>
			<td>Promega</td>
			<td>E4471</td>
			<td></td>
		</tr>
		<tr>
			<td>Supercoiled DNA Ladder</td>
			<td>New England BioLabs Inc.</td>
			<td>N0472S</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Agarose</td>
			<td>Invitrogen</td>
			<td>16500-500</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Ethidium Bromide</td>
			<td>Invitrogen</td>
			<td>15585011</td>
			<td></td>
		</tr>
		<tr>
			<td>XhoI Restriction Enzyme</td>
			<td>New England BioLabs Inc.</td>
			<td>R0146S</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>RNA extraction </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Beta-mercaptoethanol</td>
			<td>Sigma</td>
			<td>M3148-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
	</tbody>
</table>

gene expression analysis of endothelial cells exposed to shear stress using multiple parallel-plate flow chambers

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate flow chambers. Endothelial cells form the inner cellular lining of blood vessels and are chronically exposed to the frictional force of blood flow called shear stress. Under physiological conditions, endothelial cells function in the presence of various shear stress conditions. Thus, the application of shear stress conditions in in vitro models can provide greater insight into endothelial responses in vivo. The parallel-plate flow chamber previously published by Lane et al.9 is adapted to study endothelial gene regulation in the presence and absence of steady (non-pulsatile) laminar flow. Key adaptations in the set-up for laminar flow as presented here include a large, dedicated environment to house concurrent flow circuits, the monitoring of flow rates in real-time, and the inclusion of an exogenous reference RNA for the normalization of quantitative real-time PCR data. To assess multiple treatments/conditions with the application of shear stress, multiple flow circuits and pumps are used simultaneously within the same heated and humidified incubator. The flow rate of each flow circuit is measured continuously in real-time to standardize shear stress conditions throughout the experiments. Because these experiments have multiple conditions, we also use an exogenous reference RNA that is spiked-in at the time of RNA extraction for the normalization of RNA extraction and first-strand cDNA synthesis efficiencies. These steps minimize the variability between samples. This strategy is employed in our pipeline for the gene expression analysis with shear stress experiments using the parallel-plate flow chamber, but parts of this strategy, such as the exogenous reference RNA spike-in, can easily and cost-effectively be used for other applications.

Successful linearization of luciferase plasmid using restriction enzymes was confirmed by running digested products on an agarose gel (Figure 1). The size of the linearized product was confirmed using DNA ladders and by comparison with uncut plasmid.
We have adapted the parallel-plate flow chamber set-up from Lane et al.9 for experiments that require multiple conditi...

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Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers

Here, a workflow for the culture and gene expression analysis of endothelial cells under fluid shear stress is presented. Included is a physical arrangement for simultaneously housing and monitoring multiple flow chambers in a controlled environment and the use of an exogenous reference RNA for quantitative PCR.

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate ...

This method can help answer key questions in the field of endothelial biology, such as why do blood vessels have regional differences in the development of atherosclerosis? The main advantage of this technique is it allows the simultaneous study of multiple conditions under shear stress or multiple shear stress conditions. The implications of this technique extend toward understanding endothelial gene regulation in both the arterial and venous systems because various flow rates and patterns can be used.Well here, we demonstrate a system that uses steady laminar flow. The setup can be adapted for other flow patterns, including disturbed flow. Generally, individuals new to this method will struggle to maintain a continuous monolayer of cells throughout the duration of the experiment and to obtain consistent results between experiments.24 hours before the analysis, count the human endothelial cells at an early passage and seed approximately one times 10 to the sixth cells in one milliliter of medium onto one fibronectin coated glass slide per well of a four-well cell culture plate. Allow the cells to adhere to the slide for 15 minutes at 37 degrees Celsius. Then cover each slide with three milliliters of medium and place the plate in the cell culture incubator of 37 degrees Celsius and 5%CO2 for 24 hours.On the day of the experiment, fill the water tray of a large, heated, built-in environment with adjustable CO2 and heat, or beach incubator, with multiple shelves, internal electricity access, and glass doors, and confirm that adequate carbon dioxide is available for the experiment, and that the CO2 monitor is functional. To set up the flow chamber, insert a 1/8 inch female lure into one end of a number 16 soft tube, and attach a four-way stopcock to the lure. Attach the free end of the tube to the inflow side of the top plate and insert a 1/8th inch male lure into one end of a second number 16 soft tube.Attach a four-way stopcock to the male lure, and attach the free end of the tube to the outflow side of the top plate. Insert a 1/8th inch male lure and a 1/8th inch female lure into each end of a third number 16 soft tube, and attach the tubing to a bubble trap via the 1/8th inch male lure. Then attach a four-way stopcock to the other end of the tubing via the female lure.Using sterile tweezers, transfer one cell seeded glass slide to the recess on the bottom plate, cell seeded side up. Use a 10 milliliter syringe to add 10 milliliters of warm medium to the bottom plate within the red gasket line around the plate. Allowing medium to flow through the slide and cover the cells.Gently place the top plate onto the bottom plate taking care to align the sides and screw the plates together tightly. To remove air bubbles from the bubble trap, first open the inflow and bubble trap stopcocks and close the outflow stopcock. Then use a 30 milliliter syringe to gently flush 20 milliliters of warm medium through the plate.To remove bubbles from the chamber, close the bubble trap stopcock and open the outflow stopcock. Elevate the outflow side of the chamber to a 45 degree angle and use the 30 milliliter syringe to gently flush 20 milliliters of warm medium from the inflow side of the chamber. It is important to flush at an appropriate rate in the direction of the flow and to use microscope to confirm that a confluent monolayer of cells remains before exposing the cells to the laminar flow.Then close the stopcocks on both sides of the chamber and cap and confirm that the cells are still attached to the slide under a light microscope. Place the flow chamber and a previously assembled flow loop system into the beach and slowly decrease the pump speed of the flow loop assembly before pausing the pump altogether. Close the stopcocks on the flow loop system to prevent leakage and connect the chamber and loop system together via stopcocks.When all of the loops have been connected, reopen all of the stopcocks and slowly increase the pump speed while monitoring the setup for any leakage or blockage. Place the flow sensor on the chamber bridge tubing of the inflow side of the chamber with the sensor oriented in the direction of flow and adjust the pump speed to obtain the flow rate for the shear stress rate of interest. Then turn on the CO2 tank to achieve a 5%C02 concentration.Once the experimental flow period is complete, slowly decrease the peristaltic pump speed to zero and turn off the power. Quickly close all of the open stopcocks and transfer the chamber and the attached tubing with a stopcock on each side to a clean bench top. Carefully unscrew all of the screws to remove the top plate and use needle nose tweezers to transfer the glass slide from the bottom plate to a 100 millimeter diameter dish.Wash the slide in 10 milliliters of cold PBS and check the cells under a microscope to confirm cell adherence and alignment in the direction of flow. After aspirating the PBS, transfer the slide to a clean 100 millimeter diameter dish and add 350 microliters of lysis buffer supplemented with 1/100th of Beta-Mercaptoethanol from an RNA extraction kit to the slide. Use a polyethylene bladed cell scraper to scrape the cells off the slide and tilt the tissue culture dish to allow the buffer to pool at the bottom.Use forceps to remove the slide and pipette the cell lysate into a 1.5 milliliter tube on ice. Then add 10 microliters of diluted luciferase RNA to the cell lysate for downstream RNA isolation and analysis according to standard protocols. A successful linearization of the luciferase plasmid using restriction enzymes can be confirmed by running the digested products on an agarose gel and the size of the linearized product can be confirmed with DNA ladders and by comparison with an uncut plasmid.Manufacturing processes can lead to small variations in the chamber height, thus the flow rate must be calculated for each chamber to achieve the same shear stress. For experiments incorporating laminar shear stress in mammalian endothelial cells, firefly luciferase RNA can be used as an exogenous RNA spike-in. Normalization of the endogenous reference gene, the exogenous reference gene, and both endogenous and exogenous reference genes together yields similar results.Of note, in these representative experiments using two simultaneously running flow chambers with shear stress at one Pascal for each experiment, Kruppel like factor two knockdown via SI-RNA yielded a similar knockdown efficiency between the tested biological samples. While attempting this procedure it is important to remember to establish an even monolayer of cells on the slide and to profuse the flow loop system prior to attaching the flow chamber. Following this procedure, other methods, like RNA sequencing, can be used to assess genome wide RNA expression and other readouts, such as DNA and protein, can be used for further molecular analysis.After its development, this technique paved the way for researchers in the field of endothelial biology to explore the relationship between shear stress and angiogenic potential in human endothelial cells.

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Research

JoVE Journal

Bioengineering

8.9K Views.  University of Toronto. Questo metodo può aiutare a rispondere a domande chiave nel campo della biologia endoteliale, come perché i vasi sanguigni hanno differenze regionali nello sviluppo dell'aterosclerosi? Il vantaggio principale di questa tecnica è che consente lo studio simultaneo di più condizioni in condizioni di sollecitazione di taglio o più condizioni di sollecitazione di taglio. Le implicazioni di questa tecnica si estendono verso la comprensione della regolazione genica endoteliale sia nel sistema a...