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Journal of Visualized Experiments: JoVE

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https://read.qxmd.com/read/30774141/a-simple-migration-invasion-workflow-using-an-automated-live-cell-imager
#1
Xiajie Zhang, Brianna C Morten, Rodney J Scott, Kelly A Avery-Kiejda
Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option...
February 2, 2019: Journal of Visualized Experiments: JoVE
https://read.qxmd.com/read/30774136/production-of-extracellular-matrix-fibers-via-sacrificial-hollow-fiber-membrane-cell-culture
#2
Kevin Roberts, John Taehwan Kim, Shelby White, Jamie Hestekin, Jeffrey C Wolchok
Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure and healing. Extraction of extracellular matrix from fibrogenic cell cultures in vitro has potential for generation of ECM from human- and potentially patient-specific cell lines, minimizing the presence of xenogeneic epitopes which has hindered the clinical success of some existing ECM products. A significant challenge in in vitro production of ECM suitable for implantation is that ECM production by cell culture is typically of relatively low yield...
February 2, 2019: Journal of Visualized Experiments: JoVE
https://read.qxmd.com/read/30774134/short-session-high-intensity-interval-training-and-treadmill-assessment-in-aged-mice
#3
Kenneth Ladd Seldeen, Yonas Z Redae, Ramkumar Thiyagarajan, Reem Nagi Berman, Merced Marie Leiker, Bruce Robert Troen
High intensity interval training (HIIT) is emerging as a therapeutic approach to prevent, delay, or ameliorate frailty. In particular short session HIIT, with regimens less than or equal to 10 min is of particular interest as several human studies feature routines as short as a few minutes a couple times a week. However, there is a paucity of animal studies that model the impacts of short session HIIT. Here, we describe a methodology for an individually tailored and progressive short session HIIT regimen of 10 min given 3 days a week for aged mice using an inclined treadmill...
February 2, 2019: Journal of Visualized Experiments: JoVE
https://read.qxmd.com/read/30774132/modified-blood-collection-from-tail-veins-of-non-anesthetized-mice-with-a-vacuum-blood-collection-system-and-eyeglass-magnifier
#4
Xiaoyan Liu, Huanhuan Li, Jie Wu, Yijun Yu, Mingjing Zhang, Wusong Zou, Ye Gu
Blood sample collection is the basis of experimental animal research. It is of importance to obtain adequate blood samples for various research purposes. The tail veins of mice are small, and it is sometimes difficult to obtain the required blood volume by conventional puncture methods. This study investigates the superiority of repeated blood sample collection from tail veins of mice through use of a vacuum blood collection system and eyeglass magnifier (experimental group) compared to conventional blood sampling methods (conventional group), performed by beginners and experts, respectively...
February 2, 2019: Journal of Visualized Experiments: JoVE
https://read.qxmd.com/read/30774118/cereal-crop-ear-counting-in-field-conditions-using-zenithal-rgb-images
#5
José A Fernandez-Gallego, María Luisa Buchaillot, Adrian Gracia-Romero, Thomas Vatter, Omar Vergara Diaz, Nieves Aparicio Gutiérrez, María Teresa Nieto-Taladriz, Samir Kerfal, Maria Dolors Serret, José Luis Araus, Shawn C Kefauver
Ear density, or the number of ears per square meter (ears/m2 ), is a central focus in many cereal crop breeding programs, such as wheat and barley, representing an important agronomic yield component for estimating grain yield. Therefore, a quick, efficient, and standardized technique for assessing ear density would aid in improving agricultural management, providing improvements in preharvest yield predictions, or could even be used as a tool for crop breeding when it has been defined as a trait of importance...
February 2, 2019: Journal of Visualized Experiments: JoVE
https://read.qxmd.com/read/30774127/chronic-implantation-of-whole-cortical-electrocorticographic-array-in-the-common-marmoset
#6
Misako Komatsu, Takaaki Kaneko, Hideyuki Okano, Noritaka Ichinohe
Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot...
February 1, 2019: Journal of Visualized Experiments: JoVE
https://read.qxmd.com/read/30774121/an-iodide-yellow-fluorescent-protein-gap-junction-intercellular-communication-assay
#7
Joo Hye Yeo, Jinu Lee
Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and pathological roles, there is need of high-throughput screening (HTS) assays to identify GJ modulators in drug discovery and toxicology assays. A novel iodide-yellow fluorescent protein-gap junction-intercellular communication (I-YFP-GJIC) assay fulfills this need. It is a cell-based assay including acceptor and donor cells that are engineered to stably express a yellow fluorescent protein (YFP) variant, whose fluorescence is sensitively quenched by iodide, or SLC26A4, an iodide transporter, respectively...
February 1, 2019: Journal of Visualized Experiments: JoVE
https://read.qxmd.com/read/30774120/cell-based-assays-of-sineup-non-coding-rnas-that-can-specifically-enhance-mrna-translation
#8
Hazuki Takahashi, Harshita Sharma, Piero Carninci
Targeted-protein enhancement is of importance not only for the study of biological processes but also for therapeutic and biotechnological applications. Here, we present a method to selectively up-regulate protein expression of desired genes in cultured cells by means of synthetic antisense non-coding RNAs known as SINEUPs. This positive control of gene expression is at the post-transcriptional level and exerted by an inverted short interspersed nuclear element (SINE) repeat at the 3' end of SINEUPs that comprises its effector domain (ED)...
February 1, 2019: Journal of Visualized Experiments: JoVE
https://read.qxmd.com/read/30774119/generation-of-cationic-nanoliposomes-for-the-efficient-delivery-of-in-vitro-transcribed-messenger-rna
#9
Tatjana Michel, Antonia Link, Meike-Kristin Abraham, Christian Schlensak, Karlheinz Peter, Hans-Peter Wendel, Xiaowei Wang, Stefanie Krajewski
The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive properties of in vitro transcribed (IVT) mRNA. With the help of IVT mRNA, the de novo synthesis of a desired protein can be induced without changing the physiological state of the target cell. Moreover, protein biosynthesis can be precisely controlled due to the transient effect of IVT mRNA. For the efficient transfection of cells, nanoliposomes (NLps) may represent a safe and efficient delivery vehicle for therapeutic mRNA...
February 1, 2019: Journal of Visualized Experiments: JoVE
https://read.qxmd.com/read/30774135/probing-the-structure-and-dynamics-of-nucleosomes-using-atomic-force-microscopy-imaging
#10
Micah P Stumme-Diers, Tommy Stormberg, Zhiqiang Sun, Yuri L Lyubchenko
Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription to take place in eukaryotic cells. The dynamics of nucleosomes provides access to the DNA by replication and transcription machineries, and critically contributes to the molecular mechanisms underlying chromatin functions. Single-molecule studies such as atomic force microscopy (AFM) imaging have contributed significantly to our current understanding of the role of nucleosome structure and dynamics...
January 31, 2019: Journal of Visualized Experiments: JoVE
https://read.qxmd.com/read/30774131/simultaneous-electrocardiography-recording-and-invasive-blood-pressure-measurement-in-rats
#11
Hsin-Ting Lin, Yi-Lin Shiou, Wun-Jyun Jhuang, Hsiang-Chun Lee
For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. Research focusing on cardiovascular disease models, potential cardiovascular therapeutic targets or pharmaceutical agents requires assessment of systemic arterial pressure and heart rhythm changes. In situations where radio telemetry systems are not available or affordable, the technique of femoral artery cannulation is an alternative way to obtain intra-arterial pressure waveform recordings and systemic BP measurements...
January 31, 2019: Journal of Visualized Experiments: JoVE
https://read.qxmd.com/read/30774130/two-photon-imaging-of-microglial-processes-attraction-toward-atp-or-serotonin-in-acute-brain-slices
#12
Fanny Etienne, Vincenzo Mastrolia, Luc Maroteaux, Jean-Antoine Girault, Nicolas Gervasi, Anne Roumier
Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of homeostasis, undergo rapid morphological changes. For example, a laser lesion induces in a few minutes an oriented growth of microglial processes, also called "directional motility", toward the site of injury. A similar effect can be obtained by delivering locally ATP or serotonin (5-hydroxytryptamine [5-HT]). In this article, we describe a protocol to induce a directional growth of microglial processes toward a local application of ATP or 5-HT in acute brain slices of young and adult mice and to image this attraction over time by multiphoton microscopy...
January 31, 2019: Journal of Visualized Experiments: JoVE
https://read.qxmd.com/read/30774128/hybrid-printing-for-the-fabrication-of-smart-sensors
#13
Lisa-Marie Faller, Johanna Zikulnig, Matic Krivec, Ali Roshanghias, Anze Abram, Hubert Zangl
A method to combine additively manufactured substrates or foils and multilayer inkjet printing for the fabrication of sensor devices is presented. First, three substrates (acrylate, ceramics, and copper) are prepared. To determine the resulting material properties of these substrates, profilometer, contact angle, scanning electron microscope (SEM), and focused ion beam (FIB) measurements are done. The achievable printing resolution and suitable drop volume for each substrate are, then, found through the drop size tests...
January 31, 2019: Journal of Visualized Experiments: JoVE
https://read.qxmd.com/read/30774122/construction-and-use-of-an-electrical-stimulation-chamber-for-enhancing-osteogenic-differentiation-in-mesenchymal-stem-stromal-cells-in-vitro
#14
Liudmila Leppik, Mit B Bhavsar, Karla M C Oliveira, Maria Eischen-Loges, Sahba Mobini, John H Barker
Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) has been demonstrated to increase MSC osteogenic differentiation in vitro and promote bone healing in clinical settings. Here we describe the construction of an EStim cell culture chamber and its use in treating rat bone-marrow-derived MSC to enhance osteogenic differentiation. We found that treating MSCs with EStim for 7 days results in a significant increase in the osteogenic differentiation, and importantly, this pro-osteogenic effect persists long after (7 days) EStim is discontinued...
January 31, 2019: Journal of Visualized Experiments: JoVE
https://read.qxmd.com/read/30774117/ablation-of-ischemic-ventricular-tachycardia-using-a-multipolar-catheter-and-3-dimensional-mapping-system-for-high-density-electro-anatomical-reconstruction
#15
Alexandra Schratter, Georg Delle Karth, Reinhard Achleitner
Ventricular tachycardia (VT) in patients with ischemic cardiomyopathy mainly results from endocardial scars after myocardial infarction; those scars represent zones of slow conduction that allow the occurrence and maintenance of reentrant circuits. Catheter ablation enables substrate modification of those low voltage areas and thus can help to alter the scar tissue in such a way that arrhythmias cannot appear anymore. Hospitalizations of concerned patients decrease, quality of life and outcome rise. Consequently, VT ablation represents a growing field in electrophysiology, especially for patients with endocardial scars in ischemic heart disease after myocardial infarction...
January 31, 2019: Journal of Visualized Experiments: JoVE
https://read.qxmd.com/read/30774143/detection-of-endotoxin-in-nano-formulations-using-limulus-amoebocyte-lysate-lal-assays
#16
Barry W Neun, Marina A Dobrovolskaia
When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity...
January 30, 2019: Journal of Visualized Experiments: JoVE
https://read.qxmd.com/read/30774142/measuring-the-shape-and-size-of-activated-sludge-particles-immobilized-in-agar-with-an-open-source-software-pipeline
#17
Joseph E Weaver, Jon C Williams, Joel J Ducoste, Francis L de Los Reyes
Experimental bioreactors, such as those treating wastewater, contain particles whose size and shape are important parameters. For example, the size and shape of activated sludge flocs can indicate the conditions at the microscale, and also directly affect how well the sludge settles in a clarifier. Particle size and shape are both misleadingly 'simple' measurements. Many subtle issues, often unaddressed in informal protocols, can arise when sampling, imaging, and analyzing particles. Sampling methods may be biased or not provide enough statistical power...
January 30, 2019: Journal of Visualized Experiments: JoVE
https://read.qxmd.com/read/30774139/structural-design-and-manufacturing-of-a-cruiser-class-solar-vehicle
#18
Giangiacomo Minak, Tommaso M Brugo, Cristiano Fragassa, Ana Pavlovic, Felipe V de Camargo, Nicola Zavatta
Cruisers are multi-occupant solar vehicles that are conceived to compete in long-range (over 3,000 km) solar races based on the best compromise between the energy consumption and the payload. They must comply to the race's rules regarding the overall dimensions, the solar panel size, functionality, and safety and structural requirements, while the shape, the materials, the powertrain, and the mechanics are considered at the discretion of the designer. In this work, the most relevant aspects of the structural design process of a full-carbon fiber-reinforced plastic solar vehicle are detailed...
January 30, 2019: Journal of Visualized Experiments: JoVE
https://read.qxmd.com/read/30774129/a-new-straightforward-method-for-lipophilicity-logp-measurement-using-19f-nmr-spectroscopy
#19
Zhong Wang, Benjamin F Jeffries, Hannah R Felstead, Neil J Wells, Elisabetta Chiarparin, Bruno Linclau
Fluorination has become an effective tool to optimize physicochemical properties of bioactive compounds. One of the applications of fluorine introduction is to modulate the lipophilicity of the compound. In our group, we are interested in the study of the impact of fluorination on lipophilicity of aliphatic fluorohydrins and fluorinated carbohydrates. These are not UV-active, resulting in a challenging lipophilicity determination. Here, we present a straightforward method for the measurement of lipophilicity of fluorinated compounds by 19 F NMR spectroscopy...
January 30, 2019: Journal of Visualized Experiments: JoVE
https://read.qxmd.com/read/30774126/using-in-vitro-and-in-cell-shape-to-investigate-small-molecule-induced-pre-mrna-structural-changes
#20
Jingxin Wang, John Hammond, Kristen A Johnson
In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2'-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI)...
January 30, 2019: Journal of Visualized Experiments: JoVE
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