8,444 results match your criteria Journal of Visualized Experiments [Journal]


A High-content Assay for Monitoring AMPA Receptor Trafficking.

J Vis Exp 2019 Jan 28(143). Epub 2019 Jan 28.

Neuroscience Institute, Georgia State University;

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different stimuli. The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which are responsible for fast excitatory synaptic transmission in neurons, are trafficked to and from the postsynaptic surface to dynamically alter neuronal excitability. AMPA receptor trafficking is essential for synaptic plasticity and can be disrupted in neurological disease. Read More

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http://dx.doi.org/10.3791/59048DOI Listing
January 2019

Assembling Molecular Shuttles Powered by Reversibly Attached Kinesins.

J Vis Exp 2019 Jan 26(143). Epub 2019 Jan 26.

Department of Biomedical Engineering, Columbia University;

This protocol describes how to create kinesin-powered molecular shuttles with a weak and reversible attachment of the kinesins to the surface. In contrast to previous protocols, in this system, microtubules recruit kinesin motor proteins from solution and place them on a surface. The kinesins will, in turn, facilitate the gliding of the microtubules along the surface before desorbing back into the bulk solution, thus being available to be recruited again. Read More

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http://dx.doi.org/10.3791/59068DOI Listing
January 2019

Assays for the Specific Growth Rate and Cell-binding Ability of Rotavirus.

J Vis Exp 2019 Jan 28(143). Epub 2019 Jan 28.

Department of Civil and Environmental Engineering, Tohoku University; Department of Frontier Science for Advanced Environment, Graduate School of Environmental Studies, Tohoku University;

Rotavirus is the main etiological factor for infantile diarrhea. It is a double-stranded (ds) RNA virus and forms a genetically diverse population, known as quasispecies, owing to their high mutation rate. Here, we describe how to measure the specific growth rate and the cell-binding ability of rotavirus as its phenotypes. Read More

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https://www.jove.com/video/58821/assays-for-specific-growth-
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http://dx.doi.org/10.3791/58821DOI Listing
January 2019
1 Read

Shaping the Amplitude and Phase of Laser Beams by Using a Phase-only Spatial Light Modulator.

J Vis Exp 2019 Jan 28(143). Epub 2019 Jan 28.

Institut de Noves Tecnologies de la Imatge (INIT), Universitat Jaume I;

The aim of this article is to visually demonstrate the utilization of an interferometric method for encoding complex fields associated with coherent laser radiation. The method is based on the coherent sum of two uniform waves, previously encoded into a phase-only spatial light modulator (SLM) by spatial multiplexing of their phases. Here, the interference process is carried out by spatial filtering of light frequencies at the Fourier plane of certain imaging system. Read More

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http://dx.doi.org/10.3791/59158DOI Listing
January 2019

An Improved and High Throughput Respiratory Syncytial Virus (RSV) Micro-neutralization Assay.

J Vis Exp 2019 Jan 26(143). Epub 2019 Jan 26.

Murdoch Children's Research Institute; Department of Paediatrics, University of Melbourne.

Respiratory syncytial virus-specific neutralizing antibodies (RSV NAbs) are an important marker of protection against RSV. A number of different assay formats are currently in use worldwide so there is a need for an accurate and high-throughput method for measuring RSV NAbs. We describe here an imaging-based micro-neutralization assay that has been tested on RSV subgroup A and can also be adapted for RSV subgroup B and different sample types. Read More

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http://dx.doi.org/10.3791/59025DOI Listing
January 2019

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis.

J Vis Exp 2019 Jan 27(143). Epub 2019 Jan 27.

Department of Experimental Medicine, University of British Columbia; Department of Pediatrics, Division of Infectious Diseases, University of British Columbia.

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Read More

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http://dx.doi.org/10.3791/58574DOI Listing
January 2019

Immunization of Alpacas (Lama pacos) with Protein Antigens and Production of Antigen-specific Single Domain Antibodies.

J Vis Exp 2019 Jan 26(143). Epub 2019 Jan 26.

Department of Molecular and Cellular Biochemistry, University of Kentucky;

In this manuscript, a method for the immunization of alpaca and the use of molecular biology methods to produce antigen-specific single domain antibodies is described and demonstrated. Camelids, such as alpacas and llamas, have become a valuable resource for biomedical research since they produce a novel type of heavy chain-only antibody which can be used to produce single domain antibodies. Because the immune system is highly flexible, single domain antibodies can be made to many different protein antigens, and even different conformations of the antigen, with a very high degree of specificity. Read More

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http://dx.doi.org/10.3791/58471DOI Listing
January 2019

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection.

J Vis Exp 2019 Jan 26(143). Epub 2019 Jan 26.

Department of Pathology and Institute for Human Infections and Immunity, University of Texas Medical Branch.

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Read More

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http://dx.doi.org/10.3791/59095DOI Listing
January 2019
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Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal.

J Vis Exp 2019 Jan 26(143). Epub 2019 Jan 26.

School of Life Sciences, The Chinese University of Hong Kong; State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong; Key Laboratory for Regenerative Medicine, Ministry of Education, The Chinese University of Hong Kong; Centre for Novel Biomaterials, The Chinese University of Hong Kong;

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon named apoptosis reversal leads to increased tumorigenicity in various cell models under different stimuli. Previous studies have been focused on tracking this process in vitro and in vivo; however, the isolation of real reversed cells has yet to be achieved, which limits our understanding on the consequences of apoptosis reversal. Read More

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http://dx.doi.org/10.3791/58642DOI Listing
January 2019

Probiotic Studies in Neonatal Mice Using Gavage.

J Vis Exp 2019 Jan 27(143). Epub 2019 Jan 27.

Department of Epidemiology and Pediatrics, University of Nebraska Medical Centre.

Adult mouse models have been widely used to understand the mechanism behind disease progression in humans. The applicability of studies done in adult mouse models to neonatal diseases is limited. To better understand disease progression, host responses and long-term impact of interventions in neonates, a neonatal mouse model likely is a better fit. Read More

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http://dx.doi.org/10.3791/59074DOI Listing
January 2019

Multi-Modal Home Sleep Monitoring in Older Adults.

J Vis Exp 2019 Jan 26(143). Epub 2019 Jan 26.

Department of Neurology, Washington University School of Medicine; Hope Center for Neurological Disorders, Washington University School of Medicine;

The gold standard for sleep monitoring is attended in-lab polysomnography; however, this method may be cost-prohibitive and inconvenient for patients and research participants. Home sleep testing has gained momentum in the field of sleep medicine due to its convenience and lower cost, as well as being more naturalistic. The accuracy and quality of home sleep testing, however, may be variable because studies are not monitored by sleep technologists. Read More

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http://dx.doi.org/10.3791/58823DOI Listing
January 2019
1 Read

Isolation and Characterization of Human Umbilical Cord-derived Mesenchymal Stem Cells from Preterm and Term Infants.

J Vis Exp 2019 Jan 26(143). Epub 2019 Jan 26.

Department of Pediatrics, Kobe University Graduate School of Medicine;

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally isolated and characterized from bone marrow (BM), then acquired from tissues including adipose tissue, synovium, skin, dental pulp, and fetal appendages such as placenta, umbilical cord blood (UCB), and umbilical cord (UC). MSCs are a heterogeneous cell population with the capacity for (1) adherence to plastic in standard culture conditions, (2) surface marker expression of CD73/CD90/CD105/CD45/CD34/CD14/CD19/HLA-DR phenotypes, and (3) trilineage differentiation into adipocytes, osteocytes, and chondrocytes, as currently defined by the International Society for Cellular Therapy (ISCT). Read More

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http://dx.doi.org/10.3791/58806DOI Listing
January 2019

Sexual Transmission of American Trypanosomes from Males and Females to Naive Mates.

J Vis Exp 2019 Jan 27(143). Epub 2019 Jan 27.

Chagas Disease Multidisciplinary Research Laboratory, Faculty of Medicine, University of Brasilia;

American trypanosomiasis is transmitted to humans by triatomine bugs through the ingestion of contaminated food, by blood transfusions or accidently in hospitals and research laboratories. In addition, the Trypanosoma cruzi infection is transmitted congenitally from a chagasic mother to her offspring, but the male partner's contribution to in utero contamination is unknown. The findings of nests and clumps of amastigotes and of trypomastigotes in the theca cells of the ovary, in the goniablasts and in the lumen of seminiferous tubules suggest that T. Read More

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http://dx.doi.org/10.3791/57985DOI Listing
January 2019

Inducement and Evaluation of a Murine Model of Experimental Myopia.

J Vis Exp 2019 Jan 22(143). Epub 2019 Jan 22.

Department of Ophthalmology, Keio University School of Medicine;

Murine model of myopia can be a powerful tool for myopia research because of the comparatively easy genetic manipulation. One way to induce myopia in animals is to put clear minus lenses in front of eyes for weeks (lens-induced myopia, LIM). However, extant protocols for inducement and evaluation vary from laboratory to laboratory. Read More

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http://dx.doi.org/10.3791/58822DOI Listing
January 2019

Assaying for Inorganic Polyphosphate in Bacteria.

J Vis Exp 2019 Jan 21(143). Epub 2019 Jan 21.

Department of Microbiology, University of Alabama at Birmingham;

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many bacteria. There are a variety of methods for quantifying polyP in biological materials, many of which are either labor-intensive or insensitive, limiting their usefulness. We present here a streamlined method for polyP quantification in bacteria, using a silica membrane column extraction optimized for rapid processing of multiple samples, digestion of polyP with the polyP-specific exopolyphosphatase ScPPX, and detection of the resulting free phosphate with a sensitive ascorbic acid-based colorimetric assay. Read More

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http://dx.doi.org/10.3791/58818DOI Listing
January 2019
1 Read

Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats.

J Vis Exp 2019 Jan 16(143). Epub 2019 Jan 16.

Department of Anatomical Sciences and Neurobiology, University of Louisville; Kentucky Spinal Cord Injury Rehabilitation Center, University of Louisville;

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) on a treadmill is widely used as a rehabilitation tool in the SCI population with many benefits and improvements to daily life. We utilize this method of activity-based task-specific training (ABT) in rodents after SCI to both elucidate the mechanisms behind such improvements and to enhance and improve upon existing clinical rehabilitation protocols. Read More

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http://dx.doi.org/10.3791/58983DOI Listing
January 2019

Detection of Protease Activity by Fluorescent Peptide Zymography.

J Vis Exp 2019 Jan 20(143). Epub 2019 Jan 20.

Comprehensive Cancer Center, James Cancer Hospital and Solove Research Center, Ohio State University; Department of Biomedical Engineering, College of Engineering, Ohio State University;

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using electrophoresis through a resolving gel embedded with a degradable substrate. This method differs from traditional gel zymography in that a quenched fluorogenic peptide is covalently incorporated into the resolving gel instead of full length proteins, such as gelatin or casein. Read More

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http://dx.doi.org/10.3791/58938DOI Listing
January 2019
1 Read

Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation.

J Vis Exp 2019 Jan 18(143). Epub 2019 Jan 18.

Department of Basic and Clinical Sciences, Albany College of Pharmacy and Health Sciences;

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. This method utilizes hypotonic buffers, digitonin, mechanical lysis and differential centrifugation to isolate the cytoplasm, mitochondria and plasma membrane. The process can be scaled to accommodate the needs of researchers, is inexpensive and straightforward. Read More

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http://dx.doi.org/10.3791/59022DOI Listing
January 2019

In Vitro Differentiation Model of Human Normal Memory B Cells to Long-lived Plasma Cells.

J Vis Exp 2019 Jan 20(143). Epub 2019 Jan 20.

IGH, CNRS, Univ Montpellier; Laboratory for Monitoring Innovative Therapies, Department of Biological Hematology, CHU Montpellier; UFR de Médecine, Univ Montpellier;

Plasma cells (PCs) secrete large amounts of antibodies and develop from B cells that have been activated. PCs are rare cells located in the bone marrow or mucosa and ensure humoral immunity. Due to their low frequency and location, the study of PCs is difficult in human. Read More

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http://dx.doi.org/10.3791/58929DOI Listing
January 2019
1 Read

Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy.

J Vis Exp 2019 Jan 25(143). Epub 2019 Jan 25.

Department of Biomedical Engineering, School of Engineering, School of Medicine, University of Alabama at Birmingham;

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data in all cells, including stem cells, and its integrity is fundamental to its regenerative ability. Stem cells undergo rapid propagation in labs to achieve the necessary numbers for transplantation. Read More

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http://dx.doi.org/10.3791/58971DOI Listing
January 2019
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A Stainless Protocol for High Quality RNA Isolation from Laser Capture Microdissected Purkinje Cells in the Human Post-Mortem Cerebellum.

J Vis Exp 2019 Jan 17(143). Epub 2019 Jan 17.

Department of Pathology and Cell Biology, Columbia University;

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or regions from heterogenous tissues. Captured product can be used in a variety of molecular methods for protein, DNA or RNA isolation. However, preservation of RNA from postmortem human brain tissue is especially challenging. Read More

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http://dx.doi.org/10.3791/58953DOI Listing
January 2019

Development and Implementation of a Multi-Disciplinary Technology Enhanced Care Pathway for Youth and Adults with Concussion.

J Vis Exp 2019 Jan 20(143). Epub 2019 Jan 20.

Department of Biomedical Engineering, Cleveland Clinic Foundation; Concussion Center, Cleveland Clinic Foundation.

The evidence-informed standardization of care along disease lines is recommended to improve outcomes and reduce healthcare costs. The aim of this project is to 1) describe the development and implementation of the Concussion Carepath, 2) demonstrate the process of integrating technology in the form of a mobile application to enable the carepath and guide clinical decision-making, and 3) present data on the utility of the C3 app in facilitating decision-making throughout the injury recovery process. A multi-disciplinary team of experts in concussion care was formed to develop an evidence-informed algorithm, outlining best practices for the clinical management of concussion along three phases of recovery - acute, subacute, and post-concussive. Read More

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https://www.jove.com/video/58962/development-implementation-
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http://dx.doi.org/10.3791/58962DOI Listing
January 2019
3 Reads

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential.

J Vis Exp 2019 Jan 18(143). Epub 2019 Jan 18.

Insuficiencia Cardiaca y Regeneración Cardiaca (ICREC) Research Program, Health Science Research Institute Germans Trias i Pujol; Cardiology Service, Germans Trias i Pujol University Hospital; Department of Medicine, Universitat Autònoma de Barcelona; Centro de Investigación Biomédica en Red (CIBER) Cardiovascular, Instituto de Salud Carlos III.

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated researchers in the stem cell and bioengineering fields to develop in vitro high-fidelity human myocardium for both basic research and clinical applications. However, the immature phenotype of cardiac cells is a limitation on obtaining tissues that functionally mimic the adult myocardium, which is mainly characterized by mechanical and electrical signals. Read More

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http://dx.doi.org/10.3791/58934DOI Listing
January 2019

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries.

J Vis Exp 2019 Jan 22(143). Epub 2019 Jan 22.

Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore; Department of Immunobiology, Rega Institute for Medical Research, Katholieke Universiteit (KU) Leuven;

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies have been proposed and implemented for the identification of new mediators of TCR signaling, which would improve the understanding of T-cell processes, including activation and thymic selection. We describe a screening assay that enables the identification of molecules that influence TCR signaling based on the activation of developing thymocytes. Read More

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http://dx.doi.org/10.3791/58946DOI Listing
January 2019

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System.

J Vis Exp 2019 Jan 16(143). Epub 2019 Jan 16.

Department of Physics and Engineering, Doane University;

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, including Pseudomonas aeruginosa (P. aeruginosa). Here, we will describe how the system can be used to study the growth kinetics and the morphological properties such as the surface roughness and textural entropy of P. Read More

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http://dx.doi.org/10.3791/58926DOI Listing
January 2019

Monitoring Neuronal Survival via Longitudinal Fluorescence Microscopy.

J Vis Exp 2019 Jan 19(143). Epub 2019 Jan 19.

Department of Neurology, University of Michigan School of Medicine; Neuroscience Graduate Program, University of Michigan School of Medicine;

Standard cytotoxicity assays, which require the collection of lysates or fixed cells at multiple time points, have limited sensitivity and capacity to assess factors that influence neuronal fate. These assays require the observation of separate populations of cells at discrete time points. As a result, individual cells cannot be followed prospectively over time, severely limiting the ability to discriminate whether subcellular events, such as puncta formation or protein mislocalization, are pathogenic drivers of disease, homeostatic responses, or merely coincidental phenomena. Read More

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http://dx.doi.org/10.3791/59036DOI Listing
January 2019

Spatiotemporally Controlled Nuclear Translocation of Guests in Living Cells Using Caged Molecular Glues as Photoactivatable Tags.

J Vis Exp 2019 Jan 17(143). Epub 2019 Jan 17.

Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo; Riken Center for Emergent Matter Science;

The cell nucleus is one of the most important organelles as a subcellular drug-delivery target, since modulation of gene replication and expression is effective for treating various diseases. Here, we demonstrate light-triggered nuclear translocation of guests using caged molecular glue (Glue-R) tags, whose multiple guanidinium ion (Gu) pendants are protected by an anionic photocleavable group (butyrate-substituted nitroveratryloxycarbonyl; NVOC). Guests tagged with Glue-R are taken up into living cells via endocytosis and remain in endosomes. Read More

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http://dx.doi.org/10.3791/58631DOI Listing
January 2019

Probing Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices via Laser Flash Photolysis of Photoactivatable Nicotine.

J Vis Exp 2019 Jan 25(143). Epub 2019 Jan 25.

Department of Pharmacology, Northwestern University Feinberg School of Medicine;

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with subcellular location within cells where this function is carried out. To study the subcellular location of nicotinic ACh receptors (nAChRs) in native brain tissue, an optical method was developed for precise release of nicotine at discrete locations near neuronal membranes during electrophysiological recordings. Patch-clamped neurons in brain slices are filled with dye to visualize their morphology during 2-photon laser scanning microscopy, and nicotine uncaging is executed with a light flash by focusing a 405 nm laser beam near one or more cellular membranes. Read More

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http://dx.doi.org/10.3791/58873DOI Listing
January 2019

Introduction of Intracapsular Rotary-cut Procedures (IRCP): A Modified Hysteromyomectomy Procedures Facilitating Fertility Preservation.

J Vis Exp 2019 Jan 17(143). Epub 2019 Jan 17.

Department of Obstetrics and Gynecology, Peking University Shenzhen Hospital, China; Shenzhen Key Laboratory of Gynecological Diagnostic Technology Research, China;

Uterine fibroids are common benign tumors in the female reproductive system. A hysterectomy is the most effective treatment for symptomatic fibroids. For patients desiring pregnancy, laparoscopic intracapsular myomectomy (LM) is an alternative surgery option. Read More

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http://dx.doi.org/10.3791/58410DOI Listing
January 2019
1 Read

Quantification of Antibody-dependent Enhancement of the Zika Virus in Primary Human Cells.

J Vis Exp 2019 Jan 18(143). Epub 2019 Jan 18.

Department of Microbiology, Boston University School of Medicine; National Emerging Infectious Diseases Laborator, Boston University School of Medicine;

The recent emergence of the flavivirus Zika and neurological complications, such as Guillain-Barré syndrome and microcephaly in infants, has brought serious public safety concerns. Among the risk factors, antibody-dependent enhancement (ADE) poses the most significant threat, as the recent re-emergence of the Zika virus (ZIKV) is primarily in areas where the population has been exposed and is in a state of pre-immunity to other closely related flaviviruses, especially dengue virus (DENV). Here, we describe a protocol for quantifying the effect of human serum antibodies against DENV on ZIKV infection in primary human cells or cell lines. Read More

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http://dx.doi.org/10.3791/58691DOI Listing
January 2019
1 Read

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium.

J Vis Exp 2019 Jan 20(143). Epub 2019 Jan 20.

Department of Basic Sciences, Loma Linda University;

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Read More

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http://dx.doi.org/10.3791/58832DOI Listing
January 2019

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation.

J Vis Exp 2019 Jan 16(143). Epub 2019 Jan 16.

Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen;

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, they are important drug targets. We have developed a magnetic-agarose-bead-based assay platform for the investigation of proteolytic activity, which is based on the use of recombinant fusion protein substrates. In order to demonstrate the use of this assay system, a protocol is presented on the example of human immunodeficiency virus type 1 (HIV-1) protease. Read More

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http://dx.doi.org/10.3791/58824DOI Listing
January 2019
1 Read

Harvest of Endothelial Cells from the Balloon Tips of Swan-Ganz Catheters after Right Heart Catheterization.

J Vis Exp 2019 Jan 23(143). Epub 2019 Jan 23.

Cardiovascular Institute, Allegheny Health Network.

A variety of pathologies lead to pulmonary hypertension (PH), which is defined as a mean pulmonary artery pressure exceeding 25 mmHg at rest. To further diagnose and manage PH, patients undergo repeated right heart catheterizations (RHC) wherein a Swan-Ganz catheter is advanced into a branch of the pulmonary artery and a balloon is inflated to wedge the catheter tip. This article illustrates a protocol whereby pulmonary artery endothelial cells (PAECs) may be harvested from the balloon tips of Swan-Ganz catheters after RHC, and purified with an anti- CD146 affinity column technique to purify putative PAECs. Read More

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http://dx.doi.org/10.3791/58353DOI Listing
January 2019

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons.

J Vis Exp 2019 Jan 18(143). Epub 2019 Jan 18.

Department of Pediatrics, First Affiliated Hospital of Nanchang University; Center for Laboratory Medicine, First Affiliated Hospital of Nanchang University;

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms underlying sensory transmission, nociceptive regulation, and chronic pain or itch development. Implementations of electrophysiological recordings together with morphological studies based on the utility of acute spinal cord slices have further improved our understanding of neuronal properties and the composition of local circuitry in SG. Here, we present a detailed and practical guide for the preparation of spinal cord slices and show representative whole-cell recording and morphological results. Read More

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http://dx.doi.org/10.3791/58479DOI Listing
January 2019

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins.

J Vis Exp 2019 Jan 18(143). Epub 2019 Jan 18.

Department of Nephrology, Medical Faculty, Heinrich-Heine-University.

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and podocytes with their slit diaphragms. The delicate structure of the glomerular filter, especially the slit diaphragm, relies on the interplay of diverse cell surface proteins. Studying these cell surface proteins has so far been limited to in vitro studies or histologic analysis. Read More

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http://dx.doi.org/10.3791/58542DOI Listing
January 2019
1 Read

Observational Study Protocol for Repeated Clinical Examination and Critical Care Ultrasonography Within the Simple Intensive Care Studies.

J Vis Exp 2019 Jan 16(143). Epub 2019 Jan 16.

Department of Critical Care, University of Groningen, University Medical Center Groningen.

Longitudinal evaluations of critically ill patients by combinations of clinical examination, biochemical analysis and critical care ultrasonography (CCUS) may detect adverse events of interventions such as fluid overload at an early stage. The Simple Intensive Care Studies (SICS) is a research line that focuses on the prognostic and diagnostic value of combinations of clinical variables. The SICS-I specifically focused on the use of clinical variables obtained within 24 h of acute admission for prediction of cardiac output (CO) and mortality. Read More

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http://dx.doi.org/10.3791/58802DOI Listing
January 2019

Lipid Bilayer Experiments with Contact Bubble Bilayers for Patch-Clampers.

J Vis Exp 2019 Jan 16(143). Epub 2019 Jan 16.

Department of Molecular Physiology and Biophysics, University of Fukui Faculty of Medical Sciences;

Lipid bilayers provide a unique experimental platform for functional studies of ion channels, allowing the examination of channel-membrane interactions under various membrane lipid compositions. Among them, the droplet interface bilayer has gained popularity; however, the large membrane size hinders the recording of low electrical background noise. We have established a contact bubble bilayer (CBB) method that combines the benefits of planar lipid bilayer and patch-clamp methods, such as the ability to vary the lipid composition and to manipulate the bilayer mechanics, respectively. Read More

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http://dx.doi.org/10.3791/58840DOI Listing
January 2019

Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies.

J Vis Exp 2019 Jan 23(143). Epub 2019 Jan 23.

bisy e.U.; Institute of Molecular Biotechnology, Technical University of Graz;

Methanol is a well-established carbon source and inducer for efficient protein production employing Pichia pastoris (P. pastoris) as a host on micro-, lab and industrial scale. However, due to its toxicity and flammability, there is a desire to avoid methanol while maintaining the high productivity of P. Read More

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http://dx.doi.org/10.3791/58589DOI Listing
January 2019

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels.

J Vis Exp 2019 Jan 22(143). Epub 2019 Jan 22.

Department of Biomedical Engineering, The Ohio State University; The Ohio State University Comprehensive Cancer Center, Arthur G. James Cancer Hospital and Richard J. Solove Research Institute;

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. Read More

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http://dx.doi.org/10.3791/59123DOI Listing
January 2019

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination.

J Vis Exp 2019 Jan 20(143). Epub 2019 Jan 20.

Inovio Pharmaceuticals, Inc.

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, influenza, diphtheria, and viral hemorrhagic fevers. We have demonstrated that plasmid-DNA (pDNA) vaccine delivery into the skin elicits robust humoral responses in the guinea pig. However, the use of this animal to model immune responses was somewhat limited in the past due to the lack of available reagents and protocols to study T cell responses. Read More

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http://dx.doi.org/10.3791/58595DOI Listing
January 2019
3 Reads

Low-cost Protocol of Footprint Analysis and Hanging Box Test for Mice Applied the Chronic Restraint Stress.

J Vis Exp 2019 Jan 23(143). Epub 2019 Jan 23.

Division of Biology, Center for Molecular Medicine, Jichi Medical University;

Gait disturbance is frequently observed in patients with movement disorders. In mouse models used for movement disorders, gait analysis is important behavioral test to determine whether the mice mimic the symptoms of patients. Motor deficits are often induced by stress when no spontaneous motor phenotype is observed in the mouse models. Read More

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http://dx.doi.org/10.3791/59027DOI Listing
January 2019

Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens.

J Vis Exp 2019 Jan 21(143). Epub 2019 Jan 21.

Laboratory of Pathology, MedImmune;

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of tissue samples from formalin-fixed, paraffin-embedded (FFPE) biopsies has become a key tool for understanding the complexity of tumor immunology and discovering novel predictive biomarkers for cancer immunotherapy. Immunoprofiling analysis of tissues requires the evaluation of combined markers, including inflammatory cell subpopulations and immune checkpoints, in the tumor microenvironment. Read More

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http://dx.doi.org/10.3791/58390DOI Listing
January 2019
4 Reads

Handling and Assessment of Human Primary Prostate Organoid Culture.

J Vis Exp 2019 Jan 17(143). Epub 2019 Jan 17.

Department of Pathology, University of Illinois at Chicago; University of Illinois Cancer Center;

This paper describes a detailed protocol for three-dimensional (3D) culturing, handling, and evaluation of human primary prostate organoids. The process involves seeding of epithelial cells sparsely in a 3D matrix gel on a 96-well microplate with media changes to cultivate expansion into organoids. Morphology is then assessed by whole-well capturing of z-stack images. Read More

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http://dx.doi.org/10.3791/59051DOI Listing
January 2019

Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei.

J Vis Exp 2019 Jan 22(143). Epub 2019 Jan 22.

Biology Centre, Czech Academy of Science, Institute of Parasitology; Faculty of Science, University of South Bohemia;

F1-ATPase is a membrane-extrinsic catalytic subcomplex of F-type ATP synthase, an enzyme that uses the proton motive force across biological membranes to produce adenosine triphosphate (ATP). The isolation of the intact F1-ATPase from its native source is an essential prerequisite to characterize the enzyme's protein composition, kinetic parameters, and sensitivity to inhibitors. A highly pure and homogeneous F1-ATPase can be used for structural studies, which provide insight into molecular mechanisms of ATP synthesis and hydrolysis. Read More

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http://dx.doi.org/10.3791/58334DOI Listing
January 2019
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Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria.

J Vis Exp 2019 Jan 25(143). Epub 2019 Jan 25.

MRC Laboratory of Molecular Biology.

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. Read More

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https://www.jove.com/video/58981/genetic-engineering-dictyos
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http://dx.doi.org/10.3791/58981DOI Listing
January 2019
2 Reads

Spontaneous Formation and Rearrangement of Artificial Lipid Nanotube Networks as a Bottom-Up Model for Endoplasmic Reticulum.

J Vis Exp 2019 Jan 22(143). Epub 2019 Jan 22.

Centre for Molecular Medicine Norway, Faculty of Medicine, University of Oslo; Department of Chemistry, Faculty of Mathematics and Natural Sciences, University of Oslo; Department of Chemistry and Chemical Engineering, Chalmers University of Technology;

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense lipidic nanotubes that are, in terms of morphology and dynamics, reminiscent of ER. The networks are derived from phospholipid double bilayer membrane patches adhering to a transparent Al2O3 substrate. Read More

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http://dx.doi.org/10.3791/58923DOI Listing
January 2019

A Rhodopsin Transport Assay by High-Content Imaging Analysis.

J Vis Exp 2019 Jan 16(143). Epub 2019 Jan 16.

Department of Ophthalmology, University of Pittsburgh; McGowan Institute for Regenerative Medicine, University of Pittsburgh;

Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding disease that lacks effective treatment. We hypothesize that the cytotoxicity of the misfolded rhodopsin mutant can be alleviated by pharmacologically stabilizing the mutant rhodopsin protein. The P23H mutation, among the other Class II rhodopsin mutations, encodes a structurally unstable rhodopsin mutant protein that is accumulated in the endoplasmic reticulum (ER), whereas the wild type rhodopsin is transported to the plasma membrane in mammalian cells. Read More

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https://www.jove.com/video/58703/a-rhodopsin-transport-assay
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http://dx.doi.org/10.3791/58703DOI Listing
January 2019
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Generation of Cancer Cell Clones to Visualize Telomeric Repeat-containing RNA TERRA Expressed from a Single Telomere in Living Cells.

J Vis Exp 2019 Jan 17(143). Epub 2019 Jan 17.

Laboratory of Cell Biology and Molecular Genetics, Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento;

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in telomere biology, including heterochromatin formation and telomere length homeostasis. Recent findings revealed that TERRA molecules also interact with internal chromosomal regions to regulate gene expression in mouse embryonic stem (ES) cells. In line with this evidence, RNA fluorescence in situ hybridization (RNA-FISH) analyses have shown that only a subset of TERRA transcripts localize at chromosome ends. Read More

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http://dx.doi.org/10.3791/58790DOI Listing
January 2019

Determining the Likelihood of Variant Pathogenicity Using Amino Acid-level Signal-to-Noise Analysis of Genetic Variation.

J Vis Exp 2019 Jan 16(143). Epub 2019 Jan 16.

Department of Pediatrics, Division of Cardiology, Duke University School of Medicine;

Advancements in the cost and speed of next generation genetic sequencing have generated an explosion of clinical whole exome and whole genome testing. While this has led to increased identification of likely pathogenic mutations associated with genetic syndromes, it has also dramatically increased the number of incidentally found genetic variants of unknown significance (VUS). Determining the clinical significance of these variants is a major challenge for both scientists and clinicians. Read More

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http://dx.doi.org/10.3791/58907DOI Listing
January 2019

An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy.

J Vis Exp 2019 Jan 25(143). Epub 2019 Jan 25.

Research Programs Unit, Genome-Scale Biology, Biomedicum Helsinki, University of Helsinki; Department of Microbiology, Tumor, and Cell Biology (MTC), Karolinska Institutet;

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No current animal models of diabetes and oxygen-induced retinopathy develop the full-range progressive changes manifested in human proliferative diabetic retinopathy (PDR). Therefore, understanding of the disease pathogenesis and pathophysiology has relied largely on the use of histological sections and vitreous samples in approaches that only provide steady-state information on the involved pathogenic factors. Read More

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http://dx.doi.org/10.3791/59090DOI Listing
January 2019