Name: the assembly and application of 'shear rings': a novel endothelial model for orbital, unidirectional and periodic fluid flow and shear stress
Uploaded: 2016-10-31T14:00:00
Description: Watch this Scientific Journal Video about The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress at JoVE.com

The authors would like to acknowledge the assistance of Mr. Christopher Nguyen, Aaron Hunter and the Shreveport Jumpstart, SMART, and Biostart training programs as well as the Centenary College of Louisiana department of Biophysics, Shreveport, LA.

1. Construction of 150 mm Diameter Shear Rings (Figure 1)
NOTE: Shear rings may be constructed to create many different dimensions by varying the outer and inner Petri dish sizes, resulting in devices with different total surface areas, cell yields and developed ranges of shear forces. This report describes a 150 mm dish combined with an inner 100 mm dish for a total surface area of 98 cm2 (Figure 2).
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<ol>
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The construction of the shear ring system for exposing endothelial cells to shear is a simple approach to performing shear stress studies. Nevertheless, there are a few steps that are critical for obtaining superior shear rings and better results. A complete seal should be made between the inner and outer ring to prevent media from leaking which could create inconsistent shear stress among samples. If a complete seal is not made, a minimal amount of methylene chloride should be added to the edge between the inner and out...

Fluid shear stress has been shown to modulate endothelial gene programs1-5 through activation of cis-regulatory elements6, histone acetyltransferase activity7 and shear stress response elements (SSRE)8. Shear stress influences endothelial contributions towards coagulation by modulating tissue factor9 and tissue plasminogen activator (tPA)10 expression. Shear stress also influences control of angiogenesis11 and vessel remodeling by regulating PDGF-B synthesis and responsiveness8. The endothelial derived vasoactive mediators adrenomedullin, endothelin-1, urotensin II and relaxin are also regulated by shear12. Transcription of endothelial nitric oxide synthase production and nitric oxide production are both shear dependent10. Shear also controls endothelial ICAM-1 expression13. Flow-induced shear stress can therefore powerfully influence a large variety of endothelial responses. Importantly, vascular pulsations now also appear to play important roles in the pathophysiology of both normal vascular aging and forms of vascular dementia14 and may even contribute to other neurodegenerative diseases, such as multiple sclerosis15.
Venous and arterial endothelial cells are inherently exposed to diverse hemodynamic flow patterns in vivo, and many different endothelial cell phenotypes can be exhibited16. Depending on the magnitude and periodicity of flow, effects on endothelial cells may include inflammatory cell activation and apoptosis, which may reflect changes in gene or protein expression17,18. Studies on endothelial cell responses to shear phenomena therefore remain complicated by the difficulties in producing in vitro models that adequately produce such shear patterns.
Many different experimental protocols have been developed to apply fluid shear stress to endothelial cell monolayers. One of the most commonly used systems is the parallel plate flow chamber, which creates uniform laminar flow within the chamber19-21. A peristaltic pump is typically connected to create periodic flow, which can recapitulate flow characteristics typically found in many locations in vivo22. Another common set-up uses the &#39;cone and plate&#39; model, where fluid shear stress is determined by the rotational speed of the cone23. Both systems, and other arrangements similar to them, can be tedious to set up and require components that can be relatively expensive and inaccessible to many laboratories.
Another major limitation of these current models is the relatively low number of replicate studies that can be simultaneously performed, each with a relatively low surface area. This increases the time and complexity of such approaches. Therefore, an ideal model that induces unidirectional and periodic shear might be one where a high number of study replicates can be easily set up, each with a relatively large surface area. Furthermore, the aforementioned models require a fairly sophisticated setup, which may be cost-prohibitive for many users. A model that can produce fluid shear disturbances using basic laboratory materials might have several advantages.
A simple and highly economical method of applying unidirectional, periodic shear stress involves the placement of circular dishes on an orbital shaker24. This protocol is very simple and can be scaled up to achieve high numbers of study replicates, each with a relatively large surface area, as needed. However, cells located in the center of the dish are exposed to different flow patterns than cells along the periphery, yielding mixed cellular phenotypic responses in the same dish.
In this current report, we describe the construction and use of &#39;shear rings&#39;, our model for creating unidirectional and periodic shear stress. The design for the shear ring effectively limits &#39;mixed&#39; cellular shear-induced phenotypes by restricting the flow pathway within a circular culture dish to the periphery through the placement of an inner ring. The construction and operation of the shear ring is simple and economical and can be easily scaled to accommodate a wide range of orbital shakers using widely available tissue culture supplies. This model can be applied in endothelial cell experiments to provide unidirectional and periodic flow patterns within physiological and pathophysiological levels.

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">100 x 20 mm plastic tissue culture dish</td>
			<td style="width:119px;">Corning</td>
			<td align="right" style="width:119px;">430167</td>
			<td style="width:195px;">The dishes must be polystyrene</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">150 x 25 mm plastic tissue culture dish</td>
			<td style="width:119px;">Corning</td>
			<td align="right" style="width:119px;">430599</td>
			<td>The dishes must be polystyrene</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">150 mm glass petri dish&#160;</td>
			<td style="width:119px;">Fisher</td>
			<td style="width:119px;">3160150BO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15ml polystyrene tissue culture plastic tubes</td>
			<td>Falcon</td>
			<td align="right">352099</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Methylene chloride</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:119px;">D65100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">silicone rubber sealant</td>
			<td style="width:119px;">DAP</td>
			<td align="right" style="width:119px;">7079808641</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">ethanol</td>
			<td style="width:119px;">Decon</td>
			<td align="right" style="width:119px;">2701</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">3 mL transfer pipette</td>
			<td style="width:119px;">Becton-Dickinson</td>
			<td align="right" style="width:119px;">357524</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">printer paper</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">scissors</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">gloves</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">rotary tool and set</td>
			<td style="width:119px;">Dremel</td>
			<td style="width:119px;">4000-6/50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">rotary tool cutting head</td>
			<td>Dremel</td>
			<td>EZ476</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">rotary tool drill head</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">distilled water</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">orbital shaker</td>
			<td style="width:119px;">VWR</td>
			<td style="width:119px;">57018-754</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">incubator</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat retinal microvascular endothelial cells</td>
			<td>Cell Biologics</td>
			<td>RA-6065</td>
			<td></td>
		</tr>
	</tbody>
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the assembly and application of 'shear rings': a novel endothelial model for orbital, unidirectional and periodic fluid flow and shear stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

Here we present representative results from both hCMEC/D3 brain endothelial cell and rat retinal microvascular endothelial cell monolayers, cultured in shear rings.
After allowing hCMEC/D3 brain endothelial cell monolayers to grow to confluence in complete EBM, the shear rings were placed on an orbital shaker for 72 hours. Using the equation from step 3.5, the calculated maximal shear stress <img alt="Equation 6" src="/files/ftp...

Watch this Scientific Journal Video about The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress at JoVE.com

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

The overall goal of this model is to create a simple and economical method for In Vitro periodic fluid flow, and shear stress studies on endothelial cells. This method can help answer key questions in the field of Endothelial Cell Physiology. Such as, how does periodic shear affect endothelial cell shape, structure, and functioning.The main advantage to this technique is that it uses a simply assembled tissue culture device, which provides relatively large surface areas for evaluation of cellular responses. Demonstrating this procedure, will be Mr.Luke White, a medical student in my laboratory. Begin this procedure by using presentation software toe create a shear ring construction template with a 150 millimeter outer edge profile and a 100 millimeter internal diameter placed in the center of the 150 millimeter circle.Print the template on a sheet of A4 white paper. Working under a fume hood with adequate ventilation and wearing gloves pipette 10 millimeter methylene chloride into a 150 millimeter glass Petri dish. It is critical that the dish is glass as methylene chloride solubilizes most plastics.Align a 150 millimeter plastic Petri dish onto the outer shearing template. Use a rotary tool to drill a three 3 millimeter hole in the center of a 100 millimeter dish. Remove any plastic shavings produced from the drilling.While holding the 100 millimeter Petri dish with a gloved hand invert and dip the top edge of the dish into the pool of methylene chloride for approximately three seconds. Allow excess methylene chloride to drip off the 100 millimeter dish and then quickly transfer the wetted dish edge down onto the center of the 150 millimeter dish. Carefully aligning the 100 millimeter dish onto the marked template.Gently rotate the 100 millimeter dish clockwise and counterclockwise a few times to ensure good adhesion to the 150 millimeter dish. Once the methylene chloride has dried, flip the newly bound Petri dishes over and carefully inspect the points of contact. If a tight seal has not completely formed inject 0.5 milliliters of methylene chloride through the 3 millimeter hole with a transfer pipette.Pick up the dish and gently rotate it to allow methylene chloride to reach the edge. After ensuring that a tight seal has been formed between the two Petri dishes, seal off the hole in the 100 millimeter dish by applying a 3 millimeter bead of silicone rubber sealant. Using a rotary tool fitted with a flat cutting head, cut off the top 3 centimeter portion of a 15 millimeter conocole polystyrene tissue culture tube, leaving at least one centimeter of the tube below the cap.Use the flat side of the cutting head to polish the edge of the cut tube until smooth and flat. Remove any plastic shavings produced by grinding. With a marker trace the cut off 15 milliliter concole tube onto the lid of the 150 millimeter dish approximately 0.5 centimeters away from the edge of the dish.Drill a hole inside the drawn circle, leaving a margin approximately one to two millimeters from the edge of the circle. Place the cut off conocole tube top over the drilled hole. Using a transfer pitette apply approximately a quarter milliliter, or 250 microliters of methylene chloride to the cut off edges of the conocole tube to bind the conocole tube to the 150 millimeter lid.Finally, seal the 150 millimeter lid onto the 150 millimeter dish by applying a one millimeter thick bead of silicone rubber sealant around the inner edge of the top Petri dish. A schematic of an assembled sear ring is shown. The blue represents the area of plated endothelial cells.The outer an inner red arrows indicate the orbital motion of the shear ring and media inside the shear ring when placed on an orbital shaker. Prepare for the sterilization by pipetting approximately 10 millimeters of PBS into the newly formed shear ring, through the 15 milliliter conocole tube port. Swirl around to remove any debris inside the shear ring.Remove the PBS with a Pasteur pipette and vacuum aspirator. Repeat the PBS wash until all debris is removed. To sterilize the shear ring a combination of a 70%ethanol rinse and UV irradiation will be used.Unscrew the vented cap, pipette approximately 10 milliliters of 70%ethanol through the access port and screw the cap back on. Rotate and flip over the sear ring multiple times ensuring that the inside of the sear ring is thoroughly washed. Under a tissue culture hood, aspirate the excess 70%ethanol.With a spray bottle, thoroughly spray the access port and cap with 70%ethanol. Next place the shear ring and cap under UV radiation in the tissue culture hood until completely dry. Once the shear ring is dry screw the cap back on.Store the sterilized shear rings at room temperature until they are used in cell culture plating. To prepare for a shear stress study, first plate endothelial cells in sterilized shear rings following the standard cell culture protocol. Allow the culture to reach confluency prior to initiation of flow studies.When the cells have reached confluency place the experimental shear rings on an orbital shaker that is placed on the bottom shelf in the incubator to minimize shelf stress and vibration. Place static control group shear rings inside the same incubator. Leave the shear rings on the shaker for the desired duration of shear stress application.After the cell layers have been exposed to shear for the desired length of time, remove the shear rings from the incubator. Pull off the shear ring lid and retrieve the cells and or media for the desired end point analysis. Note that after opening the shear rings investigators can harvest cells and medium for analysis using several methods.Shown are representative results of the appearance of human CMEC D3 Brain Endothelial cell mono layers in shear rings following 48 hours of periodic fluid shear or static exposure it was observed that the alignment of the culture is not always parallel to the flow direction indicated by the arrows. Some responses to periodic shear are shown in Rat Retinal Microvascular Endothelial cell mono layers. 72 hours of periodic fluid shear in shear rings resulted in a significant reduction in Platelet-endothelial cell adhesion molecule relative to the Beta-actin loading control.Once mastered several Shear Rings can be constructed in about three hours if it's performed properly While attempting this procedure it's important to remember to carefully align inner dish with the outer dish, and that tight seal forms between them. After watching this video you should have a good understanding of how to construct and use shear rings as a simple method to perform In Vitro periodic fluid flow and shear stress studies on endothelial cells. Don't forget that working with methylene chloride can be hazardous and precautions, such as working under a fume hood should always be taken while performing steps that require it.

the-assembly-application-shear-rings-novel-endothelial-model-for

Research

JoVE Journal

Biology

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to elaborate distinct aspects of motor function and to determine even subtle loss of movement capacity. To enhance efficacy and resolution of testing, tests should permit qualitative and quantitative measures of motor function and be sensitive to changes in performance during recovery periods. The present study describes a new task to assess skilled walking in the rat to measure both forelimb and hindlimb function at the same time. Animals are required to walk along a horizontal ladder on which the spacing of the rungs is variable and is periodically changed. Changes in rung spacing prevent animals from learning the absolute and relative location of the rungs and so minimize the ability of the animals to compensate for impairments through learning. In addition, changing the spacing between the rungs allows the test to be used repeatedly in long-term studies. Methods are described for both quantitative and qualitative description of both fore- and hindlimb performance, including limb placing, stepping, co-ordination. Furthermore, use of compensatory strategies is indicated by missteps or compensatory steps in response to another limb&#x2019;s misplacement. 

The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein

Transient kinetic analysis is indispensable for understanding the workings of biological macromolecules, since this approach yields mechanistic information including active site concentrations and intrinsic rate constants that govern macromolecular function. In case of enzymes, for example, transient or pre-steady state measurements identify and characterize individual events in the reaction pathway, whereas steady state measurements only yield overall catalytic efficiency and specificity. Individual events such as protein-protein or protein-ligand interactions and rate-limiting conformational changes often occur in the millisecond timescale, and can be measured directly by stopped-flow and chemical-quench flow methods. Given an optical signal such as fluorescence, stopped-flow serves as a powerful and accessible tool for monitoring reaction progress from substrate binding to product release and catalytic turnover1,2.
Here, we report application of stopped-flow kinetics to probe the mechanism of action of Msh2-Msh6, a eukaryotic DNA repair protein that recognizes base-pair mismatches and insertion/deletion loops in DNA and signals mismatch repair (MMR)3-5. In doing so, Msh2-Msh6 increases the accuracy of DNA replication by three orders of magnitude (error frequency decreases from ~10-6 to10-9 bases), and thus helps preserve genomic integrity. Not surprisingly, defective human Msh2-Msh6 function is associated with hereditary non-polyposis colon cancer and other sporadic cancers6-8. In order to understand the mechanism of action of this critical DNA metabolic protein, we are probing the dynamics of Msh2-Msh6 interaction with mismatched DNA as well as the ATPase activity that fuels its actions in MMR. DNA binding is measured by rapidly mixing Msh2-Msh6 with DNA containing a 2-aminopurine (2-Ap) fluorophore adjacent to a G:T mismatch and monitoring the resulting increase in 2-aminopurine fluorescence in real time. DNA dissociation is measured by mixing pre-formed Msh2-Msh6 G:T(2-Ap) mismatch complex with unlabeled trap DNA and monitoring decrease in fluorescence over time9. Pre-steady state ATPase kinetics are measured by the change in fluorescence of 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl) coumarin)-labeled Phosphate Binding Protein (MDCC-PBP) on binding phosphate (Pi) released by Msh2-Msh6 following ATP hydrolysis9,10.
The data reveal rapid binding of Msh2-Msh6 to a G:T mismatch and formation of a long-lived Msh2-Msh6 G:T complex, which in turn results in suppression of ATP hydrolysis and stabilization of the protein in an ATP-bound form. The reaction kinetics provide clear support for the hypothesis that ATP-bound Msh2-Msh6 signals DNA repair on binding a mismatched base pair in the double helix.
F. Noah Biro and Jie Zhai contributed to this paper equally.

Msh2-Msh6 is responsible for initiating repair of replication errors in DNA. Here we present a transient kinetics approach towards understanding how this critical protein works. The report illustrates stopped-flow experiments for measuring the coupled DNA binding and ATPase kinetics underlying Msh2-Msh6 mechanism of action in DNA repair.

Transient kinetic analysis is indispensable for understanding the workings of biological macromolecules, since this approach yields mechanistic ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Introducing an Angle Adjustable Cutting Box for Analyzing Slice Shear Force in Meat

Research indicates the fibre angle of the longissimus muscle can vary, depending upon location within a steak and throughout the muscle. Instead of using the original fixed 45 &deg; or 90 &deg; cutting angle for testing shear force, a variable angle cutting box can be adjusted so the angles of the knives correspond to the fibre angle of each sample. Within 2 min after cooking to an internal temperature of 71 &deg;C on an open-hearth grill set at 210 &deg;C, a 1 cm by 5 cm core is cut from the steak, parallel to muscle fibre direction, using 2 knife blades set 1 cm apart. This warm core is then subjected to the Slice Shear Force protocol (SSF) to evaluate meat texture. The use of the variable angle cutting box and the SSF protocol provides an accurate representation of the maximal shear force, as the slice and muscle fibres are consistently parallel. Therefore, the variable angle cutting box, in conjunction with the SSF protocol, can be used as a high-throughput technique to accurately evaluate meat tenderness in different locations of the longissimus muscle and, potentially, in other muscles.

Slice shear force is a reference method for beef texture analysis. Using an angle adjustable cutting box could increase its accuracy for research purposes. The results from different locations within the longissimus muscle show a high correlation with Warner-Bratzler shear force methodology and high potential adaptability for different muscles.

Research indicates the fibre angle of the longissimus muscle can vary, depending upon location within a steak and throughout the muscle. Instead of using ...

Analysis of Oxidative Stress in Zebrafish Embryos

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

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

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

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from ...

A Uniform Shear Assay for Human Platelet and Cell Surface Receptors via Cone-plate Viscometry

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like pressure or mechanical perturbations. Mechanoreceptors detect their stimuli and transmit signals via a great diversity of mechanisms. Some of the most common roles for mechanoreceptors are in neuronal responses, like touch and pain, or hair cells which function in balance and hearing. Mechanosensation is also important for cell types which are regularly exposed to shear stress such as endothelial cells, which line blood vessels, or blood cells which experience shear in normal circulation. Viscometers are devices that detect the viscosity of fluids. Rotational viscometers may also be used to apply a known shear force to fluids. The ability of these instruments to introduce uniform shear to fluids has been exploited to study many biological fluids including blood and plasma. Viscometry may also be used to apply shear to the cells in a solution, and to test the effects of shear on specific ligand-receptor pairs. Here, we utilize cone-plate viscometry to test the effects of endogenous levels of shear stress on platelets treated with antibodies against the platelet mechanosensory receptor complex GPIb-IX.

We describe an in-solution method to apply uniform shear to platelet surface receptors using cone-plate viscometry. This method may also be used more broadly to apply shear to other cell types and cell-fragments and need not target a specific ligand-receptor pair.

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like ...