The software and technical support were courtesy of Correlated Solutions Incorporated (www.correlatedsolutions.com).

NOTE: The procedures described below were performed as part of a protocol approved by the Institutional Animal Care and Use Committee at the University of South Carolina in Columbia, South Carolina.
1. Tissue Acquisition and Dissection
<ol>
	<li>Sterilize all surgical tools prior to tissue dissection. Autoclave surgical scissors and fine standard forceps as well as surgical blades under pressure of 15 psi and temperature of 121 &#176;C for 15 min.</li>
	<li>Acquire one set of fresh porcine (7 month old Landrace males, 60-70 kg) kidneys with intact aorta from a local slaughterhouse. Transport tissue back to the laboratory in iced 1% phosphate buffered saline (PBS) solution.</li>
	<li>Immediately upon arrival, isolate the abdominal aorta from surrounding tissue using surgical scissors and forceps.</li>
	<li>Wash the vessel three times using a 50 ml syringe filled with PBS (pH 7.2). Using the scissors and forceps, remove as much perivascular tissue as possible without compromising the integrity of the sample.</li>
	<li>Vertically position a sharp razor blade onto the middle section of the vessel and make sure it is perpendicular to the vessel longitudinal axis. Create two ring samples each with a width of approximately 20 mm by applying three sequential circumferential cuts with the razor blade.</li>
	<li>Vertically position a sharp razor blade onto one ring sample such that the blade is oriented in the radial direction. Apply an acute force to yield a radial cut, which results in a strip-shaped sample for uniaxial mechanical testing. Place the sample in a 100 mm glass Petri dish and submerge in PBS until the application of surface speckling. Repeat for the second ring sample.</li>
</ol>
2. Creation of Surface Speckle Pattern
<ol>
	<li>Connect the airbrush to the pressure valve.</li>
	<li>Adjust the nozzle diameter of the airbrush to yield speckles of 60-100 &#181;m (appropriate range for nozzle diameter should be determined from preliminary studies).</li>
	<li>Pour approximately 2 ml of black tissue marking dye into the gravity feeder of the airbrush.</li>
	<li>Place the airbrush approximately 0.5 m away from the sample.</li>
	<li>Remove the sample from the Petri dish. Spray tissue marking dye on the intimal surface of the sample for approximately 5 sec under a spraying pressure of 100 psi. Repeat three times to ensure that the speckle pattern uniformly covers the sample surface.</li>
</ol>
3. Performance of Experiments
<ol>
	<li>Attach each end of the sample to a plastic strip (1 cm width x 1 cm length x 0.5 cm thickness) using a tissue adhesive. Place the sample on a tissue cutting board. Position the sample such that it lies flat and measure its dimensions using a digital caliper.</li>
	<li>Initiate system controls for mechanical testing. On the system controls home screen, select &#34;Waveform&#34; on the taskbar located on the &#34;Setup&#34; tab.</li>
	<li>Adjust the position of the upper grip of the mechanical tester to -4 mm (4 mm extension relative to the designated initial position in the system). Gently secure one plastic strip (attached to the specimen in 3.1) into the upper grip of the mechanical tester and allow the sample to hang freely. Use the digital caliper to ensure that the distance between the sample and the upper grip is less than 2 mm.</li>
	<li>Manually adjust the position of the lower grip so that the free end of the sample can be secured without extension. Gently secure the plastic strip attached to the free end of the sample into the lower grip of the mechanical tester.
	<ol>
		<li>Use the digital caliper to ensure that the distance between the sample and the lower grip is less than 2 mm. Zero the system load cell. Measure the length of the sample and use this as the reference length for calculation of global circumferential strains.</li>
	</ol>
	</li>
	<li>Enter the mechanical testing protocol. The protocol used in this demonstration entails 4 uniaxial displacement cycles which extend the sample length by 18% at a displacement rate of 0.01 mm/sec.</li>
	<li>Intermittently spray PBS on the sample throughout the remaining testing protocol to ensure that it remains hydrated.</li>
	<li>Mount the camera (5 mega pixel camera, 100 mm lens, pixel size of 3.49 &#181;m) onto a tripod which is positioned 1.5 m from the loading frame. Ensure the camera and sample surface are perpendicular by setting the camera to the lowest available field of depth and manipulating its alignment such that the entire field of view is in focus.</li>
	<li>Open the image capture software.</li>
	<li>Select &#34;PGR-2&#34; in the option &#34;Select system&#34;.</li>
	<li>Select the project path to save the images to be analyzed.</li>
	<li>Click the &#34;Time Square&#34; icon and specify the acquisition interval as 5 sec.</li>
	<li>Adjust the exposure, numerical aperture, and focus of the lens so as to get a clear view of the sample.</li>
	<li>Adjust the position of LED to provide adequate lighting on the sample.</li>
	<li>Click the &#34;Start&#34; icon in the image capture software to obtain an image of the specimen surface.</li>
	<li>Open the image analysis software.</li>
	<li>Import the image obtained. Zoom in on an individual speckle, and then count number of pixels within this individual speckle. 
	Note: Identify a representative black speckle. Define the speckle size as the linear distance between pixels on both sides of the speckle that have similar high values. For an acceptable speckle size, the number of pixels across the width of a typical speckle should be greater than 3 pixels. To improve spatial resolution in the measurements, most speckles should have no more than 5-7 pixels across the width of the speckle, whenever possible. Thus, a typical speckle for this case would range between 10 &#956;m at the smallest and 23 &#956;m at the largest in linear dimension. To determine an appropriate subset size, a typical subset should have at least 3 white and 3 black speckles across its width. If a typical speckle is 5 pixels in linear dimension, then each 31x31 subset would be at least 105 &#956;m in linear dimension. The spacing between subset centers should be at least 1/6 of the linear dimension. Thus, for a 31x31 subset size, the distance is 5 pixels which represents 18 &#956;m in linear distance.</li>
	<li>After verifying the quality of speckle pattern, simultaneously click the &#34;Run&#34; icon in the system and &#34;Start&#34; icon in the image capture software to start the test.</li>
	<li>Capture a series of images throughout testing using the camera and image capture software.</li>
</ol>
4. Clean-up Procedures after the Experiment
<ol>
	<li>Place the discarded sample in biohazard bag and close the bag. Call the Department of Environmental Health and Safety (EHS) at the University of South Carolina for proper disposal.</li>
	<li>Prepare a phosphate-free disinfectant solution with a 1:64 dilution ratio of detergent disinfectant to distilled water. Soak the surgical tools in this solution for 20 min.</li>
	<li>Thoroughly rinse the items described in 4.2 with distilled water. Dry the tools using a paper towel and then spray them with a 70% ethanol solution. Once again dry the surgical tools using a paper towel and put them back in the surgical toolbox.</li>
</ol>
5. Image Analysis to Measure the Local Strain Field
<ol>
	<li>Open the image analysis software.</li>
	<li>Click the &#34;Speckle images&#34; tab, select all images which need to be analyzed.</li>
	<li>Click on rectangle tool and select the area of interest in the first image.</li>
	<li>Enter subset size 41&#215;41 pixels and step size 5 pixels.</li>
	<li>Click start analysis tab in the software, select the interpolation as optimized 8-trap; select the criterion as zero-normalized squared differences and subset weights option as Gaussian.</li>
	<li>Set the threshold options as default in the software.</li>
	<li>Click on post-processing sub tab in start analysis tab. Click option strain computation and leave filter size and type of filter as default in software. Select tensor type Lagrange.</li>
	<li>Select data tab and then select any analyzed image for visualization of the surface strain field.</li>
</ol>

<ol>
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The authors have no potential conflicts of interest.

Although previous studies have used a wide-range of dye-tracking video methods to assess sample strain 18,20,21,23,24, our present aim is to provide a comprehensive methodology to couple uniaxial tensile testing with 2D-DIC for assessment of surface strains on vascular tissue samples. With a high resolution camera and in-house image analysis software, the strain field can be measured within a predetermined surface region as the sample undergoes uniaxial loading. Of particular relevance to mechanical testing of...

A rich history of research spanning over 50 years has been focused on quantifying the mechanical properties of vascular tissues. These studies allow us to better understand both the physiological and pathological behavior of blood vessels, provide a basis for evaluating the efficacy/compatibility of endovascular devices, and aid in the design and fabrication of engineered vascular constructs1-6. Accurate measurement of the mechanical response of soft tissues and constitutive modeling of their mechanical properties is inherently challenging due to the mechanical heterogeneity, anisotropy, and nonlinearity exhibited by most tissue types. Moreover, experimental measurements are often confounded by local complexities introduced at sample-grip interfaces in the course of mechanical testing (i.e., bending, friction, stress concentrations, tearing) and the inevitable transition of mechanical properties once tissue is excised from the living animal.
A uniaxial tensile experiment is among the simplest mechanical tests that can be performed on a specimen made of a solid material, and is often used to assess the mechanical response of vascular tissue. Results from these experiments provide useful preliminary information for both native and engineered tissue sources, and can be used to compare the effects of certain treatments, disease states, or pharmacological compounds on the mechanical behavior of the vascular wall 7-11.
Uniaxial mechanical testing of soft tissues is typically performed on samples with relatively uniform geometries, which are most commonly dog-bone or ring shaped 7,8,12-14. However, significant departure from these idealized geometries can occur due to challenges associated with tissue dissection, isolation, and clamping within the testing system. Any non-uniformity in geometry will ultimately give rise to heterogeneous stress and strain fields when the sample is subjected to uniaxial extension, with the degree of heterogeneity dependent on actual sample shape, as well as sample size (relative to the grips) and the mechanical properties of the material 9,15,16. When field heterogeneities are significant, sample strain calculations based on the relative grip positions are inaccurate and thus an insufficient basis for assessing mechanical behavior.
Video analysis systems have been widely used for strain measurements of soft tissues, often using high contrast dye markers applied to the specimen surface 17,18. Digital image correlation, an optical metrological technique which measures full-field surface strain by comparing gray level intensity values on the specimen&#39;s surface before and after deformation, has been used in conjunction with video analyses of soft tissues 19-21. There are several advantages of digital image correlation compared to interferometric methods that may be employed for measurements. First, as a non-contacting measurement technique, it minimizes the confounding effects of modifying material properties due to the way in which the measurement system affects the specimen. Second, it requires a much less stringent measurement environment and has a wider range of sensitivity and resolution than other methods. Third, endowed with the capability of capturing a full field of view, this technique can characterize both the average and the local mechanical responses. For detailed explanation of the method, readers are encouraged to see the book by Sutton 22.
To obtain strain fields on the specimen surface, a two-dimensional digital image correlation technique (2D-DIC) can be used. In short, images of the specimen are captured at unloaded and various loaded states. The first image is divided into small squares called subsets (M&#215;M pixels) which form a mesh for subsequent calculation of 2D strain fields. The position of each square in the deformed specimen is obtained using an image matching algorithm. The motion of each square is then tracked, image-by-image, yielding displacement fields which can then be used to derive deformation gradients and strains via a variety of methods, including polynomial fitting or finite element interpolation. In the present manuscript, we provide a detailed methodology for assessment of the surface strain fields on native vascular tissues via integration of uniaxial tensile testing and 2D-DIC.

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Uniaxial tensile mechanical tester</td>
			<td style="width:176px;">Enduratec</td>
			<td style="width:119px;">3230 AT/HR</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">Blue tissue marking dye</td>
			<td style="width:176px;">http://www.ebay.com/itm/Tissue-Marking-Dye-in-Bottles-2oz-Bottle-1-ea-/201193551510?pt=LH_DefaultDomain_0&#38;hash=item2ed811f696</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sprayer</td>
			<td style="width:176px;">Anest-iwata</td>
			<td>CM-B</td>
			<td>Custom Micron B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Camera</td>
			<td style="width:176px;">Point Grey</td>
			<td style="width:119px;">GS2-GE-50S5M-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lens</td>
			<td style="width:176px;">Tokina</td>
			<td style="width:119px;">AT-X M100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vascular tissue</td>
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using digital image correlation to characterize local strains on vascular tissue specimens

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and product development. Stress-strain relationships are typically obtained from mechanical testing data to enable comparative assessment among samples and in some cases identification of constitutive mechanical properties. However, errors may be introduced through the use of average strain measures, as significant heterogeneity in the strain field may result from geometrical non-uniformity of the sample and stress concentrations induced by mounting/gripping of soft tissues within the test system. When strain field heterogeneity is significant, accurate assessment of the sample mechanical response requires measurement of local strains. This study demonstrates a novel biomechanical testing protocol for calculating local surface strains using a mechanical testing device coupled with a high resolution camera and a digital image correlation technique. A series of sample surface images are acquired and then analyzed to quantify the local surface strain of a vascular tissue specimen subjected to ramped uniaxial loading. This approach can improve accuracy in experimental vascular biomechanics and has potential for broader use among other native soft tissues, engineered soft tissues, and soft hydrogel/polymeric materials. In the video, we demonstrate how to set up the system components and perform a complete experiment on native vascular tissue.

The mechanical data acquired from a ramped uniaxial extension test on vascular tissue consists of load versus applied sample displacement relationships at a given displacement rate. In this study, 2D-DIC in conjunction with uniaxial mechanical testing is used to measure the surface strain fields of the specimen in orthogonal directions at various deformed states. The viscoelastic nature of vascular tissue is manifested by the notable degree of hysteresis in the load-displacement curves pr...

Watch this Scientific Journal Video about Using Digital Image Correlation to Characterize Local Strains on Vascular Tissue Specimens at JoVE.com

Using Digital Image Correlation to Characterize Local Strains on Vascular Tissue Specimens

We describe the use of digital image correlation to characterize the local surface strain field on vascular tissue samples subjected to uniaxial tensile testing. These measurements facilitate precise quantification of the sample mechanical response and the generation of constitutive stress-strain relations.

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and ...

using-digital-image-correlation-to-characterize-local-strains-on

Research

JoVE Journal

Biology

9.4K Views.  University of South Carolina. We describe the use of digital image correlation to characterize the local surface strain field on vascular tissue samples subjected to uniaxial tensile testing. These measurements facilitate precise quantification of the sample mechanical response and the generation of constitutive stress-strain relations.

Video: Using Digital Image Correlation to Characterize Local Strains on Vascular Tissue Specimens

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Brown adipocytes have the ability to uncouple the respiratory chain in mitochondria and dissipate chemical energy as heat. Development of UCP1-positive brown adipocytes in white adipose tissues (so called beige or brite cells) is highly induced by a variety of environmental cues such as chronic cold exposure or by PPAR&gamma; agonists, therefore, this cell type has potential as a therapeutic target for obesity treatment. Although most immortalized adipocyte lines cannot recapitulate the process of "browning" of white fat in culture, primary adipocytes isolated from stromal vascular fraction in subcutaneous white adipose tissue (WAT) provide a reliable cellular system to study the molecular control of beige/brite cell development. Here we describe a protocol for effective isolation of primary preadipocytes and for inducing differentiation to beige/brite cells in culture. The browning effect can be assessed by the expression of brown fat-selective markers such as UCP1.

Primary white preadipocytes isolated from white adipose tissues in mice can be differentiated into beige/brite cells. Presented here is a reliable cellular model system to study the molecular regulation of "browning" of white fat.

Brown adipocytes have the ability to uncouple the  respiratory chain in mitochondria and dissipate chemical energy as heat.  Development of UCP1-positive ...

Easy Measurement of Diffusion Coefficients of EGFP-tagged Plasma Membrane Proteins Using k-Space Image Correlation Spectroscopy

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new insights to plasma membrane protein function and regulation. Recently, k-Space Image Correlation Spectroscopy (kICS)1&#160;was developed to enable routine measurements of diffusion coefficients directly from images of fluorescently tagged plasma membrane proteins, that avoided systematic biases introduced by probe photophysics. Although the theoretical basis for the analysis is complex, the method can be implemented by nonexperts using a freely available code to measure diffusion coefficients of proteins. kICS calculates a time correlation function from a fluorescence microscopy image stack after Fourier transformation of each image to reciprocal (k-) space. Subsequently, circular averaging, natural logarithm transform and linear fits to the correlation function yields the diffusion coefficient. This paper provides a step-by-step guide to the image analysis and measurement of diffusion coefficients via kICS.
First, a high frame rate image sequence of a fluorescently labeled plasma membrane protein is acquired using a fluorescence microscope. Then, a region of interest (ROI) avoiding intracellular organelles, moving vesicles or protruding membrane regions is selected. The ROI stack is imported into a freely available code and several defined parameters (see Method section) are set for kICS analysis. The program then generates a &#34;slope of slopes&#34; plot from the k-space time correlation functions, and the diffusion coefficient is calculated from the slope of the plot. Below is a step-by-step kICS procedure to measure the diffusion coefficient of a membrane protein using the renal water channel aquaporin-3 tagged with EGFP as a canonical example.

This paper provides a step by step guide to the fluctuation analysis technique k-Space Image Correlation Spectroscopy (kICS) for measuring diffusion coefficients of fluorescently labeled plasma membrane proteins in live mammalian cells.

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new ...

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the correct amount of protein is synthesized for the cell&#8217;s specific growth conditions. One approach for measuring mRNA decay rates is inhibiting transcription and subsequently monitoring the disappearance of the already present mRNA. The rate of mRNA decay can then be quantified, and an accurate half-life can be determined utilizing several techniques. In S. cerevisiae, protocols that measure mRNA half-lives have been developed and include inhibiting transcription of mRNA using strains that harbor a temperature sensitive allele of RNA polymerase II, rpb1-1. Other techniques for measuring mRNA half-lives include inhibiting transcription with transcriptional inhibitors such as thiolutin or 1,10-phenanthroline, or alternatively, by utilizing mRNAs that are under the control of a regulatable promoter such as the galactose inducible promoter and the TET-off system. Here, we describe measurement of S. cerevisiae mRNA decay rates using the temperature sensitive allele of RNA polymerase II. This technique can be used to measure mRNA decay rates of individual mRNAs or genome-wide.

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

The steady state level of specific mRNAs is determined by the rate of synthesis and decay of the mRNA. Genome-wide mRNA degradation rates or the decay rates of specific mRNAs can be measured by determining mRNA half-lives. This protocol focuses on measurement of mRNA decay rates in Saccharomyces cerevisiae.

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the ...

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of pre-clinical agents labeled with fluorescent probes. With recent advancements in antibody based cytotoxic drug delivery systems, understanding the alterations induced by these agents within the realm of receptor aggregation and internalization is of critical importance. This protocol leverages the well-established methodology of fluorescent immunocytochemistry and the open source FIJI distribution of ImageJ, with its inbuilt autocorrelation and image mathematical functions, to perform spatial image correlation spectroscopy (ICS). This protocol quantitates the fluorescent intensity of labeled receptors as a function of the beam area of the confocal microscope. This provides a quantitative measure of the state of target molecule aggregation on the cell surface. This methodology is focused on the characterization of static cells with potential to expand into temporal investigations of receptor aggregation. This protocol presents an accessible methodology to provide quantification of clustering events occurring at the cell surface, utilizing well established techniques and non-specialized imaging apparatus.

Antibodies that bind to target receptors on the cell surface can confer conformation and clustering alterations. These dynamic changes have implications for characterizing drug development in target cells. This protocol utilizes confocal microscopy and image correlation spectroscopy through ImageJ/FIJI to quantify the extent of receptor clustering on the cell surface.

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a 'sample preparation protocol' for maize materials (i.e., stem, leaf, and root) suitable for ordinary microcomputed tomography (micro-CT) scanning. Based on high-resolution CT images of maize stem, leaf, and root, we describe two protocols for the phenotypic analysis of vascular bundles: (1) based on the CT image of maize stem and leaf, we developed a specific image analysis pipeline to automatically extract 31 and 33 phenotypic traits of vascular bundles; (2) based on the CT image series of maize root, we set up an image processing scheme for the three-dimensional (3-D) segmentation of metaxylem vessels, and extracted two-dimensional (2-D) and 3-D phenotypic traits, such as volume, surface area of metaxylem vessels, etc. Compared with traditional manual measurement of vascular bundles of maize materials, the proposed protocols significantly improve the efficiency and accuracy of micron-scale phenotypic quantification.

We provide a novel method to improve the X-ray absorption contrast of maize tissue suitable for ordinary microcomputed tomography scanning. Based on CT images, we introduce a set of image-processing workflows for different maize materials to effectively extract microscopic phenotypes of vascular bundles of maize.

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a ...

Using En Face Immunofluorescence Staining to Observe Vascular Endothelial Cells Directly

Aberrant changes in endothelial phenotype and morphology are considered to be initial events in the pathogenesis of atherosclerosis. Direct observation of the intact endothelium will provide valuable information for understanding the cellular and molecular events in the dysfunctional endothelial cells. Here, we describe a modified en face immunofluorescence staining technique which enables scientists to obtain clear images of the intact endothelial surface and analyze the molecule expression patterns in situ. The method is simple and reliable for observing the entire endothelial monolayer at different sites of the aorta. This technique may be a promising tool for understanding the pathophysiology of atherosclerosis, especially at an early stage.

Here, we present a protocol for immunofluorescence staining to observe the endothelial cells of the mouse aorta directly. This technique is useful when studying the cellular and molecular phenotype of endothelial cells in different flow patterns and in the development of atherosclerosis.

Aberrant changes in endothelial phenotype and morphology are considered to be initial events in the pathogenesis of atherosclerosis. Direct observation of ...

Human Adipose Tissue Micro-fragmentation for Cell Phenotyping and Secretome Characterization

In the past decade, adipose tissue transplants have been widely used in plastic surgery and orthopaedics to enhance tissue repletion and/or regeneration. Accordingly, techniques for harvesting and processing human adipose tissue have evolved in order to quickly and efficiently obtain large amounts of tissue. Among these, the closed system technology represents an innovative and easy-to-use system to harvest, process, and re-inject refined fat tissue in a short time and in the same intervention (intra-operatively). Adipose tissue is collected by liposuction, washed, emulsified, rinsed and minced mechanically into cell clusters of 0.3 to 0.8 mm. Autologous transplantation of mechanically fragmented adipose tissue has shown remarkable efficacy in different therapeutic indications such as aesthetic medicine and surgery, orthopedic and general surgery. Characterization of micro-fragmented adipose tissue revealed the presence of intact small vessels within the adipocyte clusters; hence, the perivascular niche is left unperturbed. These clusters are enriched in perivascular cells (i.e., mesenchymal stem cell (MSC) ancestors) and in vitro analysis showed an increased release of growth factors and cytokines involved in tissue repair and regeneration, compared to enzymatically derived MSCs. This suggests that the superior therapeutic potential of microfragmented adipose tissue is explained by a higher frequency of presumptive MSCs and enhanced secretory activity. Whether these added pericytes directly contribute to higher growth factor and cytokine production is not known. This clinically approved procedure allows the transplantation of presumptive MSCs without the need for expansion and/or enzymatic treatment, thus bypassing the requirements of GMP guidelines, and reducing the costs for cell-based therapies.

Here, we present human adipose tissue enzyme-free micro-fragmentation using a closed system device. This new method allows the obtainment of sub-millimeter clusters of adipose tissue suitable for in vivo transplantation, in vitro culture, and further cell isolation and characterization.

In the past decade, adipose tissue transplants have been widely used in plastic surgery and orthopaedics to enhance tissue repletion and/or regeneration. ...

Divergence of Root Microbiota in Different Habitats based on Weighted Correlation Networks

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, which can effectively explore the interaction relationship or co-occurrence model of different microbial species in different environments. The purpose of this manuscript is to provide details on how to use the weighted correlation network algorithm to analyze different co-occurrence networks that may occur in microbial communities due to different ecological environments. All analysis of the experiment is performed in the WGCNA package. WGCNA is an R package for weighted correlation network analysis. The experimental data used to demonstrate these methods were microbial community data from the NCBI (National Center for Biotechnology Information) database for three niches of the rice (Oryza sativa) root system. We used the weighted correlation network algorithm to construct co-abundance networks of microbial community in each of the three niches. Then, differential co-abundance networks among endosphere, rhizoplane and rhizosphere soil were identified. In addition, the core genera in network were obtained by the "WGCNA" package, which plays an important regulated role in network functions. These methods enable researchers to analyze the response of microbial network to environmental disturbance and verify different microbial ecological response theories. The results of these methods show that the significant differential microbial networks identified in the endosphere, rhizoplane and rhizosphere soil of rice.

Network analysis was applied to evaluate the association of various ecological microbial communities, such as soil, water and rhizosphere. Presented here is a protocol on how to use the WGCNA algorithm to analyze different co-occurrence networks that may occur in the microbial communities due to different ecological environments.

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, ...

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and so on. To detect and analyze soluble or diffuse protein oligomers or aggregates, fluorescence correlation spectroscopy (FCS), which can detect the diffusion speed and brightness of a single particle with a single molecule sensitivity, has been used. However, the proper procedure and know-how for protein aggregation detection have not been widely shared. Here, we show a standard procedure of FCS measurement for diffusion properties of aggregation-prone proteins in cell lysate and live cells: ALS-associated 25 kDa carboxyl-terminal fragment of TAR DNA/RNA-binding protein 43 kDa (TDP25) and superoxide dismutase 1 (SOD1). The representative results show that a part of aggregates of green fluorescent protein (GFP)-tagged TDP25 was slightly included in the soluble fraction of murine neuroblastoma Neuro2a cell lysate. Moreover, GFP-tagged SOD1 carrying ALS-associated mutation shows a slower diffusion in live cells. Accordingly, we here introduce the procedure to detect the protein aggregation via its diffusion property using FCS.

We here introduce a procedure to measure protein oligomers and aggregation in cell lysate and live cells using fluorescence correlation spectroscopy.

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's ...