Name: visualizing scar development using scad assay - an ex-situ skin scarring assay
Uploaded: 2022-04-28T13:40:00
Description: Watch this Scientific Journal Video about Visualizing Scar Development Using SCAD Assay - An Ex-situ Skin Scarring Assay at JoVE.com

We acknowledge all the co-authors of Jiang et al. 2020 for contributing to the development of SCAD methodology13. We thank Dr. Steffen Dietzel and the Bioimaging core facility at the Ludwig-Maximilans-Universit&#228;t for access to the Multiphoton system. Y.R. was supported by the Else-Kr&#246;ner-Fresenius-Stiftung (2016_A21), the European Research Council Consolidator Grant (ERC-CoG 819933), and the LEO Foundation (LF-OC-21-000835).

The model presented below provides a detailed step-by-step description of the generation of SCAD assay as briefly described in Jiang et al., 202013. SCAD sample preparations were performed after sacrificing the animals as per the international and the Government of Upper Bavaria guidelines. Animals were housed at the animal facility of the Helmholtz Centre Munich. The rooms were maintained with optimal humidity and constant temperature with a 12 h light cycle. Animals were supplied with food and w...

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Several models have already been developed to understand scar formation after injury. While a lot of advances have been rendered in this regard but, actual mechanisms are still not clear. In contrast to the previous technique, the SCAD model incorporates all cell types and cutaneous layers, thereby maintaining the complexity of native skin18,19. This methodology is capable of generating fundamental datasets that are important in understanding molecular mechanisms...

Wound healing is a process of restoration of breached wounds. Tissue injuries in invertebrates result in partial or complete regeneration. In contrast, mammals respond to deep injury by scarring, a process tailored to quickly seal wounds with dense plugs of matrix fibers that minimize the breached area and at the same time permanently deform the injured site1,2,3. Large skin burns or deep open wounds in mammals result in pathological phenotypes such as hypertrophic or keloid scars4,5. These exuberant scars cause a tremendous burden on clinical and global healthcare systems. In the US alone, scar management costs about $10 billion annually6,7. Therefore, the development of relevant methodologies are required to better understand the fundemental processes and mechanisms involved in scar formation.
In recent years, a wide range of studies in mice has revealed heterogeneous fibroblast populations with distinct functional potencies based on their origins in certain skin locations8,9,10. In back skin, Rinkevich et al., 2015, identified that a specific fibroblast population with an early embryonic expression of Engrailed-1 (En1), termed EPF (Engrailed positive fibroblast) contributes to cutaneous scarring upon wounding. Conversely,&#160; another fibroblast lineage with no history of engrailed expression, Engrailed negative fibroblast (ENF), does not contribute to scar formation8. Fate mapping of these En1 lineages using Cre-driven transgenic mouse lines crossed to fluorescence reporter mouse lines such as R26mTmG (En1Cre x R26mTmG) allows visualization of EPF and ENF populations.
Studying fibroblast migration in vivo over several days is limited by ethical and technical constraints. Furthermore, compound, viral and neutralizing antibody library screens to modulate pathways involved in scarring is technically challenging. Previously used in vitro or ex vivo models lack the ability to visualize fibroblast migration and scar formation in genuine skin microenvironments, uniformity in scar development, as well as tissue complexity that emulates in vivo skin environments11,12. To overcome the above limitations, we developed an ex vivo scarring assay termed SCAD (SCar-like tissue in A Dish)13,14. This simple assay can be performed by excising 2 mm full-thickness skin containing the epidermis, the dermis, and the subcutaneous fascia regions and culturing them in serum-supplemented DMSO media for up to 5 days. Scars generated from SCAD reliably replicate transcriptomic and proteomic hallmarks of in vivo scars. In addition, SCADs generated from relevant transgenic mouse lines (e.g., En1 mice) crossed with fluorescent reporter mouse lines allow the visualization of fibroblast migration dynamics and scar development at an unprecedented resolution. Furthermore, this model can be easily adapted for any high throughput applications (e.g., compound library, antibody library, or viral screening)13,14. In this article, we describe an optimized protocol to generate SCADs and subsequent downstream processing applications to study cellular and matrix dynamics in scar development.

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			<td>10% Tween 20, Nonionic Detergent</td>
			<td>Biorad Laboratories</td>
			<td>1610781</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin, Cold ethanol fract</td>
			<td>Sigma</td>
			<td>A4503-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12, HEPES, no phenol red-500 mL</td>
			<td>LIFE Technologies</td>
			<td>11039021</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, no calcium, no magnesium</td>
			<td>Gibco</td>
			<td>14190169</td>
			<td></td>
		</tr>
		<tr>
			<td>Epredia Cryostar NX70 Cryostat</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Epredia SuperFrost Plus Adhesion slides</td>
			<td>Fisher scientific</td>
			<td>J1800AMNZ</td>
			<td>Adhesion slides</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, qualified, heat inactivated, E.U.-approved, South America Origin-500 mL</td>
			<td>LIFE Technologies</td>
			<td>&#160;10500064</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G with DAPI</td>
			<td>Life Technologies</td>
			<td>00 4959 52</td>
			<td>Mounting medium with DAPI</td>
		</tr>
		<tr>
			<td>Forceps curved with fine points with guidepinstainless steel(tweezers)125 mm length</td>
			<td>Fisher Scientific</td>
			<td>12381369</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin from porcine skin</td>
			<td>Sigma</td>
			<td>G2500-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement-100 mL</td>
			<td>LIFE Technologies</td>
			<td>35050038</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS, calcium, magnesium, no phenol red-500 mL</td>
			<td>LIFE Technologies</td>
			<td>14025092</td>
			<td></td>
		</tr>
		<tr>
			<td>Ibidi Gas incubation system for CO2 and O2</td>
			<td>Ibidi</td>
			<td>11922</td>
			<td></td>
		</tr>
		<tr>
			<td>Ibidi Heating system</td>
			<td>Ibidi</td>
			<td>10915</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica SP8 upright microscope - Multiphoton excitation 680&#8211;1300 nm</td>
			<td>Leica</td>
			<td></td>
			<td>Equipped with a 25x water-dipping objective (HC IRAPO L 25x/1.00&#8201;W) in combination with a tunable laser (Spectra-Physics, InSight DS&#8201;+&#8201;Single)</td>
		</tr>
		<tr>
			<td>Non Essential Amino Acids</td>
			<td>LIFE Technologies</td>
			<td>11140035</td>
			<td></td>
		</tr>
		<tr>
			<td>NuSieve GTG Agarose ,25 g</td>
			<td>Biozym /Lonza</td>
			<td>859081</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT Embedding Matrix</td>
			<td>Carlroth</td>
			<td>6478.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde, 16% W/V AQ. 10 x10 mL</td>
			<td>VWR International</td>
			<td>43368.9M</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen-Strep</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Stiefel Biopsy-Punch 2 mm</td>
			<td>Stiefel</td>
			<td>270130</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight Sharp/Sharp Dissecting Scissors 11.4 cm</td>
			<td>Fisher Scientific</td>
			<td>15654444</td>
			<td></td>
		</tr>
		<tr>
			<td>Thimerosal Bioxtra, 97%&#8211;101%</td>
			<td>Sigma-Aldrich</td>
			<td>T8784-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Axioimager M2 upright microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
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visualizing scar development using scad assay - an ex-situ skin scarring assay

The mammalian global response to sealing deep tissue wounds is through scar formation and tissue contraction, mediated by specialized fascia fibroblasts. Despite the clinical significance of scar formation and impaired wound healing, our understanding of fascia fibroblast dynamics in wound healing is cursory due to the lack of relevant assays that enable direct visualization of fibroblast choreography and dynamics in complex environments such as in skin wounds. This paper presents a protocol to generate ex- situ skin scars using SCAD or &#34;SCar-like tissue in A Dish&#34; that emulate the complex environment of skin wounds. In this assay, 2 mm full-thickness skin is excised and cultured upside down in media for 5 days, during which scars and skin contractures develop uniformly. This methodology, coupled with fibroblast-lineage specific transgenic mouse models, enables visualization of individual fibroblast lineages across the entire wound repair process. Overall, this protocol aids&#160;researchers in understanding fundamental processes and mechanisms of wound repair,&#160;directly exploring the effects of modulators on wound healing outcomes.

Generation of SCADs can be separated into three essential steps: Harvesting&#160;back skin from P0-P1 mice, generating of full-thickness biopsy punches, and subsequent culture of individual scads up to 5 days in 96-well plates. As a readout, this assay can further be applied to analyze the spatial and temporal aspects of scarring. The spatial analysis utilizes 2D and 3D immune-labeling of tissues to study spatial localization of cellular and matrix components within developing scar tissue. Spatiotemporal studies allow vi...

Watch this Scientific Journal Video about Visualizing Scar Development Using SCAD Assay - An Ex-situ Skin Scarring Assay at JoVE.com

Visualizing Scar Development Using SCAD Assay - An Ex-situ Skin Scarring Assay

This protocol describes the generation of a skin-fascia explant termed "SCar like tissue in A Dish" or SCAD. This model allows unprecedented visualization of single fibroblasts during scar formation.

Visualizing Scar Development Using SCAD Assay - An Ex-situ Skin Scarring Assay

The mammalian global response to sealing deep tissue wounds is through scar formation and tissue contraction, mediated by specialized fascia fibroblasts. ...

Previously used in vitro or ex vivo models lack dermal cell components and complexity of skin tissue. This ex situ SCAD assay overcomes a set limitation and enables studying the development of scars outside of wounded animal. The SCAD assay allows visualization of fibroblast migration and scar formation in skin microenvironments.It enables screening libraries of activators or inhibitors to understand the mechanistic basis of scar formation. High-throughput screening assays of flat libraries using the SCAD model in understanding fundamental process and the molecule course involved in wound repair. The SCAD assay is easy to perform using the skin explants.For consistent scar, it is recommended to select post-natal day zero or day one skin. After sacrificing post-natal day zero or day one newborn pup, use a sterile surgical scalpel to carefully excise 1.5-by-1.5-centimeter full thickness dorsal back skin till the skeletal muscle layer. Peel the skin using sterile curved forceps, ensuring that the superficial fascia is intact with the underlying panniculus carnosus muscle.Wash the excised tissue with 50 to 100 milliliters of cold DMEM F-12 medium to remove contaminating blood. Then, wash the tissue with Hanks balanced salt solution to maintain tissue and cell viability. Place the skin upside down with superficial fascia on top in a 10-centimeter Petri dish containing DMEM F-12 medium.Then, using a disposable two-millimeter biopsy punch, excise full-thickness round skin pieces to generate scabbed tissues, ensuring that superficial fascia is intact with underlying panniculus carnosus muscle until the epidermis. Add 200 microliters of fresh DMEM F-12 complete medium without phenol red into each well of a 96-well plate. Using sterile forceps, transfer and fully submerge individual scabbed tissue upside down to the wells of a 96-well plate.Transfer the plate to a cell culture incubator maintained under standard conditions. On days two and four of culture, remove the medium leaving 10 microliters in the well and add fresh pre-warmed DMEM F-12 complete medium along with the treatment compounds to maintain continued cell and tissue viability conditions. For live imaging of SCADs, prepare 30 milliliters of 2 to 3%low-melting agarose solution in PBS in a glass bottle by heating in a microwave.After boiling, immediately transfer the bottle and cool the liquid agarose solution in a 40-degrees-Celsius water bath. Next, transfer the SCAD tissue with fascia or scar facing upward onto the center of a 35-millimeter dish. Then embed the SCAD at room temperature by slowly pouring 40-degrees-Celsius liquid agarose onto the tissue using a 1000-microliter pipette tip.Agarose polymerizes within two minutes. After polymerization, add two milliliters of pre-warmed DMEM F-12 complete medium. Then using a confocal or a multiphoton microscope equipped with suitable incubation systems explained in the manuscript, acquire time-lapse images of day zero to day one.For tissue harvesting, wash the tissues at relevant time points by replacing the media with sterile PBS. Using sterile forceps, transfer each SCAD to a 1.5-milliliter microcentrifuge tube containing 500 microliters of 2%paraformaldehyde to fix the tissues overnight at four degrees Celsius. The next day, wash the tissues three times with PBS before proceeding to two-or three-dimensional immunofluorescence staining as described in the text manuscript.The representative whole-mount bright-field images of SCADs on days zero and five are shown here. The Masson's trichrome staining of vertical tissue sections reveals signatures of tissue scarring, tissue contraction, and accumulation of extracellular matrix at the scar core at days zero and five. The immunofluorescence staining of SCADs at days zero and five are shown.From early and late stage scar, the ingrained positive fibroblast is shown in green and the ingrained negative fibroblast in red. For three-dimensional imaging, tissues were embedded in agarose solution and topped with PBSGT. The representative images demonstrate exclusive localization of N-cadherin protein at the scar site of a day-five SCAD.The three-dimensional time lapse imaging setup equipped with an incubation chamber displayed early events of progression of the fibroblast swarms over the first 12 hours or early stages of scar development. The graphical representation of single-cell tracked cellular trajectories of individual fibroblasts over scar development is presented here. It's crucial to place the SCAD tissue upside down inside the well plate so the fascia faces upwards.Failure to do this would result in unavoidable variations in migration patterns. This procedure allows to examine dermal layers for treatments or culture using chemical modulators, neutralizing antibodies, or viral methods. This helps in the assessment of pathological fibrotic responses across diverse medical settings.We showed the expression of connexin-43 and N-cadherin as key molecules involved in fascia mobilization upon wounding. The SCAD methodology allows the identification of novel therapeutic strategies to curtail scarring and fibrosis.

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Research

JoVE Journal

Biology

Titration of Human Coronaviruses Using an Immunoperoxidase Assay

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for viruses that do not cause significant cytopathic effects, which is the case for prototype strains 229E and OC43 of human coronavirus (HCoV). Therefore, an alternative indirect immunoperoxidase assay (IPA) was developed for the detection and titration of these viruses and is described herein. Susceptible cells are inoculated with serial logarithmic dilutions of virus-containing samples in a 96-well plate format. After viral growth, viral detection by IPA yields the infectious virus titer, expressed as 'Tissue Culture Infectious Dose 50 percent' (TCID50). This represents the dilution of a virus-containing sample at which half of a series of laboratory wells contain infectious replicating virus. This technique provides a reliable method for the titration of HCoV-229E and HCoV-OC43 in biological samples such as cells, tissues and fluids. This article is based on work first reported in Methods in Molecular Biology (2008) volume 454, pages 93-102.

In this video, we demonstrate an alternative method for detection and titering of viruses using an enzymatic antigen detection technique known as an immunoperoxidase assay. Here, we will show you how to collect your viral samples, prepare the cells for testing, and finally the immunoperoxidase assay using serial dilutions to determine the viral titer.

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for ...

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 ...

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results.
In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample.
Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.)
In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders.
We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity.

An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life ...

Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and on target membranes (t-SNAREs) catalyze intracellular vesicle fusion1-4. Reconstitution assays are essential for dissecting the mechanism and regulation of SNARE-mediated membrane fusion5. In a cell fusion assay6,7, SNARE proteins are expressed ectopically at the cell surface. These "flipped" SNARE proteins drive cell-cell fusion, demonstrating that SNAREs are sufficient to fuse cellular membranes. Because the cell fusion assay is based on microscopic analysis, it is less efficient when used to analyze multiple v- and t-SNARE interactions quantitatively.
Here we describe a new assay8 that quantifies SNARE-mediated cell fusion events by activated expression of &beta;-galactosidase. Two components of the Tet-Off gene expression system9 are used as a readout system: the tetracycline-controlled transactivator (tTA) and a reporter plasmid that encodes the LacZ gene under control of the tetracycline-response element (TRE-LacZ). We transfect tTA into COS-7 cells that express flipped v-SNARE proteins at the cell surface (v-cells) and transfect TRE-LacZ into COS-7 cells that express flipped t-SNARE proteins at the cell surface (t-cells). SNARE-dependent fusion of the v- and t-cells results in the binding of tTA to TRE, the transcriptional activation of LacZ and expression of &beta;-galactosidase. The activity of &beta;-galactosidase is quantified using a colorimetric method by absorbance at 420 nm.
The vesicle-associated membrane proteins (VAMPs) are v-SNAREs that reside in various post-Golgi vesicular compartments10-15. By expressing VAMPs 1, 3, 4, 5, 7 and 8 at the same level, we compare their membrane fusion activities using the enzymatic cell fusion assay. Based on spectrometric measurement, this assay offers a quantitative approach for analyzing SNARE-mediated membrane fusion and for high-throughput studies.

We have developed a cell fusion assay that quantifies SNARE-mediated membrane fusion events by activated expression of &beta;-galactosidase.

The interactions of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)  proteins on vesicles (v-SNAREs) and on target membranes ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest.

Provided is a protocol for developing a real-time recombinase polymerase amplification assay to quantify initial concentration of DNA samples using either a thermal cycler or a microscope and stage heater. Also described is the development of an internal positive control. Scripts are provided for processing raw real-time fluorescence data.

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to ...

G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group developed an intra-molecular FRET sensor to detect the interaction between G&#945; subunits and GPCRs in live cells following agonist stimulation. Here, we detail the protocol for detecting changes in FRET between the &#946;2-adrenergic receptor and the G&#945;s C-terminus peptide upon treatment with 100 &#181;M isoproterenol hydrochloride as previously characterized1. Our FRET sensor is a single polypeptide consisting serially of a full-length GPCR, a FRET acceptor fluorophore (mCitrine), an ER/K SPASM (systematic protein affinity strength modulation) linker, a FRET donor fluorophore (mCerulean), and a G&#945; C-terminal peptide. This protocol will detail cell preparation, transfection conditions, equipment setup, assay execution, and data analysis. This experimental design detects small changes in FRET indicative of protein-protein interactions, and can also be used to compare the strength of interaction across ligands and GPCR-G protein pairings. To enhance the signal-to-noise in our measurements, this protocol requires heightened precision in all steps, and is presented here to enable reproducible execution.

Simple methods to detect the selective activation of G proteins by G protein-coupled receptors remain an outstanding challenge in cell signaling. Here, F&#1255;rster resonance energy transfer (FRET) biosensors have been developed by pairwise tethering a GPCR to G protein peptides to probe conformational changes at controlled concentrations in live cells.

F&#1255;rster resonance energy transfer (FRET)-based studies have become increasingly common in the investigation of GPCR signaling. Our research group ...

Evaluating Vascular Hyperpermeability-inducing Agents in the Skin with the Miles Assay

The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby the permeability of the endothelium to blood cells, plasma macromolecules, and water can be adapted according to the physiological need. In certain diseases, cytokines and growth factors are released that target the endothelial barrier to transiently increase vascular permeability; however, their prolonged presence may cause chronic vascular hyperpermeability and thereby tissue-damaging edema. The Miles assay is an in vivo technique that allows researchers to study vascular hyperpermeability through the proxy measurement of vascular leakage. Here, we provide a detailed protocol on how to perform this procedure in the mouse, which is the most widely used model organism to study mammalian physiology and pathology. The procedure involves the intravenous injection of Evans blue dye to label the circulating albumin followed by multiple intradermal injections of permeability-inducing agents and vehicle control solutions into opposing flanks of the mouse. Consequently, Evans blue dye gradually leaks into the dermis, where it accumulates and can be extracted for quantification as leakage induced by the permeability-inducing agent relative to the vehicle. The Miles assay can be performed in wild type or genetically modified mouse models and may be combined with drug administration to study molecular mechanisms that regulate vascular permeability and identify agents/targets capable of inducing or blocking hyperpermeability.

Here, we present a protocol to measure the vascular leakage induced by intradermal administration of permeability promoting agents into the murine skin. This technique can be used to determine the ability of molecules to promote or inhibit vascular leakage or to study the molecular mechanisms that regulate vascular permeability.

The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby ...