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National Institutes of Health

153 ARTICLES PUBLISHED IN JoVE

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Neuroscience

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology
Arvydas Maminishkis 1, Sheldon S. Miller 1
1National Eye Institute, National Institutes of Health

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

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Immunology and Infection

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites
Sittiporn Pattaradilokrat 1, Jian Li 1,2, Xin-zhuan Su 1
1National Institute of Allergy and Infectious Diseases, National Institutes of Health, 2School of Life Science, Xiamen University

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

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Biology

Computer-assisted Large-scale Visualization and Quantification of Pancreatic Islet Mass, Size Distribution and Architecture
Abraham Kim 1, German Kilimnik 1, Charles Guo 1, Joshua Sung 1, Junghyo Jo 2, Vipul Periwal 2, Piotr Witkowski 3, Philip Dilorio 4, Manami Hara 1
1Department of Medicine, University of Chicago, 2Laboratory of Biological Modeling, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 3Department of Surgery, University of Chicago, 4Diabetes Division, University of Massachusetts

Novel computer-assisted methods of large-scale procurement and analysis of immunohistochemically stained pancreatic specimens are described: (1) Virtual Slice capture of the entire section; (2) Mass analysis of large-scale data; (3) Reconstruction of 2D Virtual Slices; (4) 3D islet mapping; and (5) Mathematical analysis.

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Neuroscience

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons
Chan-Ying Zheng 1, Ronald S. Petralia 1, Ya-Xian Wang 1, Bechara Kachar 1
1National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda

FRAP has been used to quantify the mobility of Green Fluorescence Protein (GFP)-tagged proteins in cultured cells. We examined the mobile/immobile fractions of the GFP by analyzing the fluorescence recovery percentage after photobleaching. In this study, FRAP was performed at spines of hippocampal neurons.

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Biology

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo
Wenling Li 1, Yoh-suke Mukouyama 1
1Laboratory of Stem Cell and Neuro-Vascular Biology, Genetics and Developmental Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

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Immunology and Infection

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging
Joel R. Meyerson 1,2, Tommi A. White 1, Donald Bliss 3, Amy Moran 3, Alberto Bartesaghi 1, Mario J. Borgnia 1, M. Jason V. de la Cruz 1, David Schauder 1, Lisa M. Hartnell 1, Rachna Nandwani 1,4, Moez Dawood 5, Brianna Kim 6, Jun Hong Kim 7, John Sununu 8, Lisa Yang 9, Siddhant Bhatia 10, Carolyn Subramaniam 1, Darrell E. Hurt 11, Laurent Gaudreault 12, Sriram Subramaniam 1
1Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 2The Medical Research Council Mitochondrial Biology Unit, University of Cambridge , 3National Library of Medicine, National Institutes of Health, 4Massachusetts Institute of Technology, 5William Fremd High School, 6University of Virginia , 7Duke University , 8Yale University, 9University of Notre Dame , 10Washington University in St. Louis , 11Bioinformatics and Computational Biosciences Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12Thomas Jefferson High School for Science and Technology

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

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Immunology and Infection

DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat (VNTR) - Fragment Length Analysis (FLA)
Ronald W. Jensen 1, Jason Rivest 1, Wei Li 1, Varalakshmi Vissa 1
1Department of Microbiology, Immunology and Pathology, Colorado State University

Leprosy, caused by Mycobacterium leprae, is still endemic in many places. In order to learn about the spread and mode of transmission of leprosy, it is important to determine which strain of M. leprae has infected a patient. Variable numbers of tandem repeats (VNTR) typing is one such method.

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Bioengineering

Simultaneous Synthesis of Single-walled Carbon Nanotubes and Graphene in a Magnetically-enhanced Arc Plasma
Jian Li 1, Alexey Shashurin 1, Madhusudhan Kundrapu 1, Michael Keidar 1
1Department of Mechanical and Aerospace Engineering, The George Washington University

Anodic arc discharge is one of the most practical and efficient methods to synthesize various carbon nanostructures. To increase the arc controllability and flexibility, a non-uniform magnetic field was introduced to process the one-step synthesis of large-scale graphene flakes and high-purity single-walled carbon nanotubes.

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Neuroscience

Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection
Carlene S. Brandon 1, Christina Voelkel-Johnson 2, Lindsey A. May 3, Lisa L. Cunningham 3
1Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 2Department of Microbiology & Immunology, Medical University of South Carolina, 3National Institute on Deafness and Other Communication Disorders, National Institutes of Health

Mechanosensory hair cells are the receptor cells of the inner ear. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice. We present the dissection of the adult mouse utricle, and we demonstrate adenovirus-mediated infection of supporting cells in cultured utricles.

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Immunology and Infection

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers
Kathleen C. Prins *1,2, Gaia Vasiliver-Shamis *2,3, Michael Cammer 1,2, David Depoil 1,2, Michael L. Dustin 2, Catarina E. Hioe 1,4
1Department of Pathology, New York University Langone School of Medicine, 2Program in Molecular Pathogenesis, Marty and Helen Kimmel Center for Biology and Medicine and Skirball Institute for Biomolecular Medicine, 3Laboratory of Molecular Immunogenetics, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, 4Veteran Affairs New York Harbor Healthcare System

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

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Medicine

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies
Wei Li 1, Shu Zhu 1, Yusong Zhang 1, Jianhua Li 1, Andrew E. Sama 1, Ping Wang 1, Haichao Wang 1
1The Feinstein Institute for Medical Research, North Shore – LIJ Health System

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

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Neuroscience

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain
Maria Chiara G. Monaco 1, Dragan Maric 2, Alexandra Bandeian 1, Emily Leibovitch 1, Wan Yang 1, Eugene O. Major 1
1Laboratory of Molecular Medicine and Neuroscience, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 2Laboratory of Neurophysiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

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Biology

Isolating Primary Melanocyte-like Cells from the Mouse Heart
Hayoung Hwang 1, Fang Liu 1, Mark D. Levin 1, Vickas V. Patel 1
1Penn Cardiovascular Institute, Perelman School of Medicine, University of Pennsylvania

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. 

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Biology

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection
Abrahim I. Orabi 1, Kamaldeen A. Muili 1, Dong Wang 1, Shunqian Jin 1, George Perides 2, Sohail Z. Husain 1
1Rangos Research Center, Pediatric Gastroenterology, Hepatology, and Nutrition, Children's Hospital of Pittsburgh of UPMC, 2Department of Surgery, Tufts University Medical Center

We describe a reproducible method of preparing mouse pancreatic acinar cells from a mouse for the purpose of examining acinar cell calcium signals and cellular injury with physiologically and pathologically relevant stimuli. A method for adenoviral infection of these cells is also provided.

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Biology

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins
Andrius Masedunskas 1,2, Natalie Porat-Shliom 1, Muhibullah Tora 1, Oleg Milberg 1,3, Roberto Weigert 1
1Intracellular Membrane Trafficking Unit, Oral and Pharyngeal Cancer Branch National Institute of Dental and Craniofacial Research, National Institutes of Health, 2Department of Biology, University of North Carolina at Chapel Hill , 3Department of Chemical & Biochemical Engineering and Department of Biomedical Engineering, Rutgers University

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

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Medicine

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung
Yan Cai 1, Shioko Kimura 1
1Laboratory of Metabolism, National Cancer Institute, National Institutes of Health

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented.

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Behavior

Simultaneous Scalp Electroencephalography (EEG), Electromyography (EMG), and Whole-body Segmental Inertial Recording for Multi-modal Neural Decoding
Thomas C. Bulea 1,2, Atilla Kilicarslan 2, Recep Ozdemir 2,3,4, William H. Paloski 3,4, Jose L. Contreras-Vidal 2,4,5
1Functional and Applied Biomechanics Group, National Institutes of Health, 2Laboratory for Non-invasive Brain-Machine Interface Systems, Department of Electrical and Computer Engineering, University of Houston, 3Department of Health and Human Performance, University of Houston, 4Center for Neuromotor & Biomechanics Research, University of Houston, 5Department of Biomedical Engineering, University of Houston

Development of an effective brain-machine-interface (BMI) system for restoration and rehabilitation of bipedal locomotion requires accurate decoding of user's intent. Here we present a novel experimental protocol and data collection technique for simultaneous non-invasive acquisition of neural activity, muscle activity, and whole-body kinematics during various locomotion tasks and conditions.

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Neuroscience

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury
Paul Lu 1,2, Lori Graham 2, Yaozhi Wang 2, Di Wu 2, Mark Tuszynski 1,2
1Veterans Administration Medical Center, San Diego, 2Department of Neurosciences, University of California, San Diego

Fibrin matrices containing growth factors were used to retain grafted neural stem cells into sites of complete spinal cord transection. Grafted cells completely filled the lesion cavity and differentiated into multiple neural cell types, including neurons that extended axons into host spinal cord over long distances.

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Neuroscience

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents
Annalisa Scimemi 1, Jeffrey S. Diamond 1
1Synaptic Physiology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

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Medicine

Dual-phase Cone-beam Computed Tomography to See, Reach, and Treat Hepatocellular Carcinoma during Drug-eluting Beads Transarterial Chemo-embolization
Vania Tacher 1, MingDe Lin 2, Nikhil Bhagat 1, Nadine Abi Jaoudeh 3, Alessandro Radaelli 4, Niels Noordhoek 4, Bart Carelsen 4, Bradford J. Wood 3, Jean-François Geschwind 1
1Russell H. Morgan Department of Radiology and Radiological Science, Division of Vascular and Interventional Radiology, The Johns Hopkins Hospital, 2Clinical Informatics, Interventional, and Translational Solutions (CIITS), Philips Research North America, 3Center for Interventional Oncology, Interventional Radiology Section, National Institutes of Health, 4Interventional X-ray, Philips Healthcare

Dual-phase cone-beam computed tomography (DP-CBCT) is a useful intraprocedural imaging technique for transarterial chemo-embolization treatment with drug-eluting beads of hepatocellular carcinoma. DP-CBCT has been used to perform three major steps in oncologic interventional radiology: tumor localization (see), navigation and intraprocedural catheter guidance (reach), and intraprocedural evaluation of treatment success (treat).

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Neuroscience

Measurement of Total Calcium in Neurons by Electron Probe X-ray Microanalysis
Natalia B. Pivovarova 1, S. Brian Andrews 1
1Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health

This paper describes the application of cryoanalytical electron microscopy to the quantitative measurement of total calcium content and distribution at subcellular resolution in physiologically defined biological specimens.

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Immunology and Infection

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking
Olga Pavlova 1, Raffaele Ieva 1, Harris D Bernstein 1
1Genetics and Biochemistry Branch of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution “snapshots” of an ordered assembly pathway in a living cell.

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JoVE Journal

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis
Kevin G. Chen 1, Rebecca S. Hamilton 1, Pamela G. Robey 2, Barbara S. Mallon 1
1NIH Stem Cell Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 2Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

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Biology

SIVQ-LCM Protocol for the ArcturusXT Instrument
Jason D. Hipp *1, Jerome Cheng *2, Jeffrey C. Hanson *1, Avi Z. Rosenberg 1, Michael R. Emmert-Buck 1, Michael A. Tangrea 1, Ulysses J. Balis 2
1Laboratory of Pathology, National Cancer Institute, National Institutes of Health, 2Department of Pathology, University of Michigan

SIVQ-LCM is an innovative approach that harnesses a computer algorithm, Spatially Invariant Vector Quantization (SIVQ), to drive the laser capture microdissection (LCM) process. The SIVQ-LCM workflow greatly improves the speed and accuracy of microdissection, with applications in both the research and clinical settings.

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Neuroscience

Live Imaging of Drosophila Larval Neuroblasts
Dorothy A. Lerit 1, Karen M. Plevock 1, Nasser M. Rusan 1
1National Heart, Lung, and Blood Institute, National Institutes of Health

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

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Medicine

Substernal Thyroid Biopsy Using Endobronchial Ultrasound-guided Transbronchial Needle Aspiration
Abhishek Kumar 1, Arjun Mohan 1, Samjot S. Dhillon 2, Kassem Harris 2
1Division of Pulmonary and Critical Care Medicine, State University of New York, Buffalo, 2Interventional Pulmonology Section, Division of Medicine, Roswell Park Cancer Institute, State University of New York, Buffalo

Substernal thyroid lesions are common, and need to be differentiated from malignancy. Obtaining percutaneous fine needle biopsy is not possible due to its retrosternal location. This article proposes a protocol for biopsy of substernal thyroid lesions using Endobronchial Ultrasound-guided Transbronchial Needle Aspiration (EBUS-TBNA).

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Medicine

Catheterization of the Carotid Artery and Jugular Vein to Perform Hemodynamic Measures, Infusions and Blood Sampling in a Conscious Rat Model
Jing Feng 1, Yvonne Fitz 1, Yan Li 1, Melinda Fernandez 1, Irene Cortes Puch 1, Dong Wang 1, Stephanie Pazniokas 2, Brandon Bucher 3, Xizhong Cui 1, Steven B. Solomon 1
1Critical Care Medicine Department, Clinical Center, National Institutes of Health, 2Harvard Apparatus, 3ADInstruments

Vascular accesses to measure hemodynamics, provide fluids and perform blood sampling are important to any small animal model study. We present a technique for implanting catheters into the carotid artery and the common jugular vein in an anesthetized rat for connecting to a system to perform monitoring, infusions and sampling.

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Developmental Biology

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells
Tongguang Wang 1, Elliot Choi 1, Maria Chiara G. Monaco 2, Eugene O. Major 2, Marie Medynets 1, Avindra Nath 1
1Translational Neuroscience Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 2Laboratory of Molecular Medicine and Neuroscience, National Institute of Neurological Disorders and Stroke, National Institutes of Health

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

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Behavior

Functional Near Infrared Spectroscopy of the Sensory and Motor Brain Regions with Simultaneous Kinematic and EMG Monitoring During Motor Tasks
Theresa Sukal-Moulton 1, Ana Carolina de Campos 1, Christopher J. Stanley 1, Diane L. Damiano 1
1Functional and Applied Biomechanics Section, Rehabilitation Medicine Department, Clinical Center, National Institutes of Health

Monitoring brain activity during upright motor tasks is of great value when investigating the neural source of movement disorders. Here, we demonstrate a protocol that combines functional near infrared spectroscopy with continuous monitoring of muscle and kinematic activity during 4 types of motor tasks.

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Developmental Biology

Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor
Trevor Cerbini 1, Yongquan Luo 1, Mahendra S. Rao 2, Jizhong Zou 1
1National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, 2Q Therapeutics

TALEN-mediated gene editing at the safe harbor AAVS1 locus enables high-efficiency transgene addition in human iPSCs. This protocol describes the procedures for preparing iPSCs for TALEN and donor vector delivery, transfecting iPSCs, and selecting and isolating iPSC clones to achieve targeted integration of a GFP gene to generate reporter lines.

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Medicine

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay
Christopher J. Johnson 1, Christina M. Carlson 2, Aaron R. Morawski 3, Alyson Manthei 4, Neil R. Cashman 5
1USGS National Wildlife Health Center, 2Department of Soil Science, University of Wisconsin–Madison, 3Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 4Merial Veterinary Scholars Program, School of Veterinary Medicine, University of Wisconsin–Madison, 5Department of Neurology, University of British Columbia

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

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Biology

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification
Nathan P. Manes 1, Jessica M. Mann 1, Aleksandra Nita-Lazar 1
1Cellular Networks Proteomics Unit, Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

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Medicine

A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine
Mitsuhiro Yoneda 1, Alfredo A. Molinolo 2, Jerrold M. Ward 3, Shioko Kimura 1, Robert A. Goodlad 4
1National Cancer Institute, Laboratory of Metabolism, National Institutes of Health, 2National Institute of Dental and Craniofacial Research, Oral and Pharyngeal Cancer Branch, National Institutes of Health, 3Global VetPathology, 4Centre for Pathology, Hammersmith Hospital, Imperial College

The use of a simple device to cut and ‘roll’ mouse intestines to rapidly prepare whole mount preparations is described.

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Biology

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP
Mark J. Henderson *1, Emily S. Wires *1, Kathleen A. Trychta 1, Xiaokang Yan 1, Brandon K. Harvey 1
1National Institute on Drug Abuse, National Institutes of Health

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

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JoVE Journal

From Constructs to Crystals – Towards Structure Determination of β-barrel Outer Membrane Proteins
Nicholas Noinaj 1, Stephen Mayclin 2, Ann M. Stanley 2,3, Christine C. Jao 2,3, Susan K. Buchanan 2
1Department of Biological Sciences, Markey Center for Structural Biology, Purdue University, 2National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health, 3National Institute of General Medical Sciences (NIGMS), National Institutes of Health

β-barrel outer membrane proteins (OMPs) serve many functions within the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Here, we hope to alleviate a known bottleneck in structural studies by presenting protocols for the production of β-barrel OMPs in sufficient quantities for structure determination by X-ray crystallography or NMR spectroscopy.

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Behavior

A Method for Evaluating the Reinforcing Properties of Ethanol in Rats without Water Deprivation, Saccharin Fading or Extended Access Training
Eric Augier 1, Russell S. Dulman 2, Erick Singley 2, Markus Heilig 1
1Center for Social and Affective Neuroscience, IKE, Linköping University, 2Laboratory of Clinical and Translational Studies, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health

This protocol describes a novel and efficient method to quickly initiate operant responding for ethanol in rats that, contrary to standard methods, does not require water deprivation or saccharin/sucrose fading to initiate responding.

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Medicine

Apical Resection Mouse Model to Study Early Mammalian Heart Regeneration
Jianhua Xiong 1, Jian Hou 2
1Center for Molecular Medicine, National Heart, Lung, and Blood Institute, National Institutes of Health, 2State Key Laboratory for Agrobiotechnology, College of Biological Science, China Agricultural University

A step-by-step video protocol of apical resection is demonstrated in this study. Apical resection is a recently highlighted surgical approach in mammalian heart regeneration research. This study may promote the application of apical resection as a standard methodology in research into the mechanism underlying heart regeneration.

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Neuroscience

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses
Jun Zhang 1, Ronald S. Petralia 2, Ya-Xian Wang 2, Jeffrey S. Diamond 1
1Synaptic Physiology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 2Advanced Imaging Core, National Institute on Deafness and Other Communication Disorders, National Institutes of Health

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

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JoVE Core

SSVEP-based Experimental Procedure for Brain-Robot Interaction with Humanoid Robots
Jing Zhao 1, Wei Li 1,2, Xiaoqian Mao 1, Mengfan Li 1
1School of Electrical Engineering and Automation, Tianjin University, 2Department of Computer & Electrical Engineering and Computer Science, California State University

The overall goal of this method is to establish an SSVEP-based experimental procedure by integrating multiple software programs to enable the study of brain-robot interaction with humanoid robots, which is prospective in assisting the sick and elderly as well as performing unsanitary or dangerous jobs.

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Engineering

Using Synchrotron Radiation Microtomography to Investigate Multi-scale Three-dimensional Microelectronic Packages
Holly D. Carlton 1, John W. Elmer 1, Yan Li 2, Mario Pacheco 2, Deepak Goyal 2, Dilworth Y. Parkinson 3, Alastair A. MacDowell 3
1Materials Engineering Division, Lawrence Livermore National Laboratory, 2Assembly Test and Technology Development Failure Analysis Labs, Intel Corporation, 3Advanced Light Source, Lawrence Berkeley National Laboratory

For this study synchrotron radiation micro-tomography, a non-destructive three-dimensional imaging technique, is employed to investigate an entire microelectronic package with a cross-sectional area of 16 x 16 mm. Due to the synchrotron's high flux and brightness the sample was imaged in just 3 min with an 8.7 µm spatial resolution.

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Biology

Maintenance of a Drosophila melanogaster Population Cage
Juan Manuel Caravaca 1, Elissa P. Lei 1
1Laboratory of Cellular and Developmental Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health

This manuscript reports a detailed protocol for culturing, on a regular basis, a population of Drosophila melanogaster using a fly population cage.

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Medicine

A Step by Step Protocol for Subretinal Surgery in Rabbits
Sami Al-Nawaiseh 1, Fabian Thieltges 1, Zengping Liu 1,2, Claudine Strack 1, Ralf Brinken 1, Norbert Braun 3, Marc Wolschendorf 3, Arvydas Maminishkis 5, Nicole Eter 4, Boris V. Stanzel 1,6
1Department of Ophthalmology, University of Bonn, 2Department of Ophthalmology, National University of Singapore, 3Geuder AG, 4Department of Ophthalmology, University of Münster, 5Section on Epithelial and Retinal Physiology and Disease, National Eye Institute/National Institutes of Health, 6Surgical Retina Department, Singapore National Eye Centre

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

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Medicine

Cutaneous Surgical Denervation: A Method for Testing the Requirement for Nerves in Mouse Models of Skin Disease
Shelby C. Peterson 1, Isaac Brownell *2, Sunny Y. Wong *1
1Dermatology, Cell and Developmental Biology, University of Michigan, 2Dermatology Branch, National Cancer Institute, National Institutes of Health

This article includes detailed protocols for genetic labeling of mouse skin, surgical denervation, skin biopsy and visualizing labeled epithelia by whole-mount β-galactosidase staining. These methods can be used to test the requirement for nerves in mouse models of normal and pathological skin.

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Behavior

The Treadmill Fatigue Test: A Simple, High-throughput Assay of Fatigue-like Behavior for the Mouse
John P. Dougherty 1, Danielle A. Springer 2, Marvin C. Gershengorn 1
1Laboratory of Endocrinology and Receptor Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 2Murine Phenotyping Core, National Heart, Lung, and Blood Institute, National Institutes of Health

Fatigue is a common, undertreated and frequently poorly-understood symptom in many diseases and disorders. New preclinical assays of fatigue may help to improve current understanding and future treatment of fatigue. To that end, the current protocol provides a novel means of measuring fatigue-like behavior in the mouse.

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Immunology and Infection

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages
Xueting Jin 1, Howard S. Kruth 1
1Experimental Atherosclerosis Section, National Heart, Lung, and Blood Institute, National Institutes of Health

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

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Medicine

Ferric Chloride-induced Murine Thrombosis Models
Wei Li 1,2, Marvin Nieman 3, Anirban Sen Gupta 4
1Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, 2Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, 3Department of Pharmacology, Case Western Reserve University, 4Department of Biomedical Engineering, Case Western Reserve University

We report a refined procedure of the ferric chloride (FeCl3)-induced thrombosis models on carotid and mesenteric artery as well as vein, characterized efficiently using intravital microscopy to monitor time to occlusive thrombi formation.

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Immunology and Infection

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice
Jennifer H. Madenspacher 1, Michael B. Fessler 1
1Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

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Genetics

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining
Zhe Chen 1, Hong Xu 1
1Lab of Molecular Genetics, National Heart, Lung and Blood Institute, National Institutes of Health

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2´-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

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Medicine

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency
Dong Wang 1,2,3,4, Grigol Tediashvili 1,2,3, Simon Pecha 4, Hermann Reichenspurner 4, Tobias Deuse 1,2,3,4, Sonja Schrepfer 1,2,3,4
1Transplant and Stem Cell Immunobiology Lab, University Heart Center Hamburg, 2Department of Surgery, Transplant and Stem Cell Immunobiology Lab, University of California San Francisco (UCSF), 3Cardiovascular Research Center (CVRC) and DZHK German Center for Cardiovascular Research), partner site Hamburg/Kiel/Luebeck, 4Cardiovascular Surgery, University Heart Center Hamburg

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

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JoVE Journal

Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method
Barbora Piknova 1, Ji Won Park 1, Katelyn S. Cassel 1, Cameron N. Gilliard 1, Alan N. Schechter 1
1Molecular Medicine Branch, NIDDK, NIH

Nitric oxide (NO) is an important signaling molecule in vascular homeostasis. NO production in vivo is too low for direct measurement. Chemiluminescence provides useful insight into NO cycle via measuring its precursors and oxidation products, nitrite and nitrate. Nitrite / nitrate determination in body tissues and fluids is explained.

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JoVE Core

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes
Javier Traba *1, Pietro Miozzo *2, Billur Akkaya 3, Susan K. Pierce 2, Munir Akkaya 2
1Laboratory of Mitochondrial Biology and Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, 2Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 3Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health

The study of metabolism is becoming increasingly relevant to immunological research. Here, we present an optimized method for measuring glycolysis and mitochondrial respiration in mouse splenocytes, and T and B lymphocytes.

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Medicine

Two Techniques to Create Hypoparathyroid Mice: Parathyroidectomy Using GFP Glands and Diphtheria-Toxin-Mediated Parathyroid Ablation
Ruiye Bi 1,2, Yi Fan 2,3, En Luo 2,4, Quan Yuan 2,4, Michael Mannstadt 1
1Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, 2West China School of Stomatology, Sichuan University, 3Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, 4State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University

Mice with acquired hypoparathyroidism would be useful for studying novel drug therapies for hypoparathyroidism. Two procedures to create such mice are demonstrated. The GFP-PTX mouse is generated by surgical parathyroidectomy guided by green fluorescing parathyroid glands. A second, non-surgical approach is based on parathyroid-specific expression of the diphtheria toxin receptor.

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Immunology and Infection

Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles
Anush Arakelyan 1, Wendy Fitzgerald 1, Sonia Zicari 1, Murad Vagida 2, Jean-Charles Grivel 3, Leonid Margolis 1
1Section on Intercellular Interactions, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, 2Laboratory of Atherothrombosis, Cardiology Department, Moscow State University of Medicine and Dentistry, 3Sidra Medical and Research Center

Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles coupled to antibodies recognizing surface antigens. The captured virions or vesicles were labeled with fluorescent antibodies against other surface antigens. The resultant complexes were separated in high magnetic field and analyzed with conventional flow cytometers triggered on fluorescence.

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Cancer Research

A Mouse Model of Fatigue Induced by Peripheral Irradiation
Brian S. Wolff 1, Michael A. Renner 1, Danielle A. Springer 2, Leorey N. Saligan 1
1Symptom Biology Unit, National Institute of Nursing Research, National Institutes of Health, 2Murine Phenotyping Core, National Heart, Lung, and Blood Institute, National Institutes of Health

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

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Bioengineering

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair
Jian Li 1, Holger Jahr 2,3, Wei Zheng 4, Pei-Gen Ren 1
1Center for Translational Medicine Research and Development, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, 2Department of Orthopedic Surgery, Maastricht UMC+, 3Department of Orthopaedic Surgery, University Hospital RWTH, 4Research Laboratory for Biomedical Optics and Molecular Imaging, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

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Genetics

Single Molecule Analysis of Laser Localized Psoralen Adducts
Jing Huang 1, Himabindu Gali 1, Julia Gichimu 1, Marina A. Bellani 1, Durga Pokharel 1, Manikandan Paramasivam 1, Michael M. Seidman 1
1Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health

Lasers are frequently used in studies of the cellular response to DNA damage. However, they generate lesions whose spacing, frequency, and collisions with replication forks are rarely characterized. Here, we describe an approach that enables the determination of these parameters with laser localized interstrand crosslinks.

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Genetics

Systemic Delivery of MicroRNA Using Recombinant Adeno-associated Virus Serotype 9 to Treat Neuromuscular Diseases in Rodents
Naemeh Pourshafie 1, Philip R. Lee 2, Ke-lian Chen 1, George G. Harmison 1, Laura C. Bott 1,3, Kenneth H. Fischbeck 1, Carlo Rinaldi 1,4
1Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 2Section on Nervous System Development and Plasticity, The Eunice Kennedy Shriver National Institute of Child and Human Development, National Institutes of Health, 3Department of Molecular Biosciences, Rice Institute for Biomedical Research, Northwestern University, 4Department of Physiology, Anatomy and Genetics, University of Oxford

Here we describe the delivery of microRNA using a recombinant adeno-associated virus serotype 9 in a mouse model of a neuromuscular disease. A single peripheral administration in mice resulted in sustained miRNA overexpression in muscle and motor neurons, providing an opportunity to study miRNA function and therapeutic potential in vivo.

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Immunology and Infection

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM
Jason Yi 1, Asit Manna 1, Valarie A. Barr 1, Jennifer Hong 2, Keir C. Neuman 2, Lawrence E. Samelson 1
1Laboratory of Cellular & Molecular Biology, National Cancer Institute, National Institutes of Health, 2Laboratory of Single Molecule Biophysics, National Heart, Lung, and Blood, Institute, National Institutes of Health

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.

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Medicine

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients
Elena K. Schneider 1, Felisa Reyes-Ortega 1, Jian Li 1,2, Tony Velkov 1
1Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 2Monash Biomedicine Discovery Institute, Department of Microbiology, Monash University

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

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Medicine

A Chronic Cardiac Ischemia Model in Swine Using an Ameroid Constrictor
Karen J. Keeran *1, Kenneth R. Jeffries 1, Arthur D. Zetts 1, Joni Taylor 1, Shawn Kozlov 1, Timothy J. Hunt *1
1Animal Surgery and Resources Core, National Heart, Lung, and Blood Institute, National Institutes of Health

The purpose of this protocol is to demonstrate the placement of a delayed constricting device (an ameroid constrictor) around a coronary artery in a swine model. This device creates an ischemic area of the heart that is useful for studying new diagnostic imaging techniques and new methods of treatment.

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Neuroscience

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay
Wei Li 1,2, Chen Zhang 1, Xiulian Sun 3
1Department of Neurology, Qilu Hospital of Shandong University, 2Department of Neurology, Qingdao Municipal Hospital, 3Brain Research Institute, Qilu Hospital of Shandong University

This protocol aims to describe a method to examine the Ca2+ retention capacity and Ca2+- triggered mitochondrial swelling of isolated mitochondria of SH-SY5Y cells step-by-step.

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Biology

Assessing Urinary Tract Junction Obstruction Defects by Methylene Blue Dye Injection
Kangsun Yun 1
1Cancer and Developmental Biology Laboratory, National Cancer Institute, National Institutes of Health

Methylene blue dye injection into the renal pelvis facilitates the assessment of urinary tract junction obstruction defects during mouse embryonic urinary tract development. Here, a protocol for methylene blue dye injection into the renal pelvis is described.

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Bioengineering

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System
Anjali Nandal 1,2, Barbara Mallon 3, Bhanu P. Telugu 1,2,4
1Department of Animal and Avian Sciences, University of Maryland, 2Animal Bioscience and Biotechnology Laboratory, ARS, USDA, 3NIH Stem Cell Unit, Bethesda, National Institutes of Health, 4RenOVAte Biosciences Inc

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

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Medicine

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice
Evandro F. Fang 1,6, Konstantinos Palikaras 2, Nuo Sun 3, Elayne M. Fivenson 1, Ryan D. Spangler 4, Jesse S. Kerr 1, Stephanie A. Cordonnier 1, Yujun Hou 1, Eszter Dombi 5, Henok Kassahun 6, Nektarios Tavernarakis 2,7, Joanna Poulton 5, Hilde Nilsen 6, Vilhelm A. Bohr 1,8
1Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, 2Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology - Hellas, 3Center for Molecular Medicine, National Heart Lung and Blood Institute, National Institutes of Health, 4Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, 5Nuffield Department of Obstetrics and Gynaecology, University of Oxford, 6Department of Clinical Molecular Biology, University of Oslo and Akershus University Hospital, 7Department of Basic Sciences, Faculty of Medicine, University of Crete, 8Danish Center for Healthy Aging, University of Copenhagen

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

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Cancer Research

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay
Michael M. Lizardo 1,2, Poul H. Sorensen 2,3
1Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 2BC Cancer Agency, Provincial Health Services Authority, 3Department of Pathology and Laboratory Medicine, University of British Columbia

The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

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Genetics

Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome
Vid Leko 1,2, Smitha Sripathy 1, Robin L. Adrianse 1, Taylor Loe 1, Angela Park 1, Uyen Lao 1, Eric J. Foss 1, Marisa S. Bartolomei 3, Antonio Bedalov 1,4
1Clinical Research Division, Fred Hutchinson Cancer Research Center, 2Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, 3Epigenetics Program, Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, 4Departments of Medicine and Biochemistry, University of Washington

We report a small hairpin RNA (shRNA) and next generation sequencing-based protocol for identifying regulators of X-chromosome inactivation in a murine cell line with firefly luciferase and hygromycin resistance genes fused to the methyl CpG binding protein 2 (MeCP2) gene on the inactive X chromosome.

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Cancer Research

Using the Dot Assay to Analyze Migration of Cell Sheets
Christina H. Stuelten 1
1Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health

Cell migration is essential for development, tissue maintenance and repair, and tumorigenesis, and is regulated by growth factors, chemokines, and cytokines. This protocol describes the dot assay, a two-dimensional, unconstrained migration assay to assess the migratory phenotype of attached, cohesive cell sheets in response to microenvironmental cues.

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Medicine

Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta
Grigol Tediashvili 1,2,3, Dong Wang 1,2,3,4, Hermann Reichenspurner 4, Tobias Deuse 1,2,3,4, Sonja Schrepfer 1,2,3,4
1Transplant and Stem Cell Immunobiology Lab, University Heart Center, 2Department of Surgery, Transplant and Stem Cell Immunobiology Lab, University of California San Francisco (UCSF), 3Cardiovascular Research Center (CVRC) and DZHK German Center for Cardiovascular Research, 4Cardiovascular Surgery, University Heart Center

This article demonstrates a murine model to study the development of myointimal hyperplasia (MH) after aortic balloon injury.

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Medicine

Posterior Semicircular Canal Approach for Inner Ear Gene Delivery in Neonatal Mouse
Kevin Isgrig 1, Wade W. Chien 1,2
1National Institute on Deafness and Other Communication Disorders, National Institutes of Health, 2Department of Otolaryngology-Head & Neck Surgery, Johns Hopkins School of Medicine

In this study, we describe the posterior semicircular canal approach as a reliable method for inner ear gene delivery in neonatal mice. We show that gene delivery through the posterior semicircular canal is able to perfuse the entire inner ear.

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Immunology and Infection

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence
Riti Sharan *1, Hee-Jeong Yang *2, Preeti Sule 1, Jeffrey D. Cirillo 1
1Department of Microbial Pathogenesis and Immunology, Texas A&M Health Science Center, 2Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

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Developmental Biology

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations
Yuho Kim 1,2,3, Jonathan M. Memme 1,2, David A. Hood 1,2
1Muscle Health Research Centre, York University, 2School of Kinesiology and Health Science, York University, 3National Heart, Lung, and Blood Institute, National Institutes of Health

This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

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Immunology and Infection

Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry
Nermi L. Parrow *1, Pierre-Christian Violet *2, Hongbin Tu 2, James Nichols 3, Corinne A. Pittman 4, Courtney Fitzhugh 4, Robert E. Fleming 1,5, Narla Mohandas 6, John F. Tisdale 3, Mark Levine 2
1Department of Pediatrics, Saint Louis University School of Medicine, 2Molecular and Clinical Nutrition Section, Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, 3Molecular and Clinical Hematology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, 4Sickle Cell Branch, National Heart, Lung and Blood Institute, National Institutes of Health, 5Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, 6Red Cell Physiology Laboratory, New York Blood Center

Here we present techniques to measure red cell deformability and cellular heterogeneity by ektacytometry. These techniques are applicable to general investigations of red cell deformability and specific investigations of blood diseases characterized by the presence of both rigid and deformable red cells in circulation, such as sickle cell anemia.

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Biochemistry

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae
Yu-Hua Lo 1, Monica C. Pillon 1, Robin E. Stanley 1
1Signal Transduction Laboratory, National Institutes of Environmental Health Sciences, Department of Health and Human Services, National Institutes of Health

This method describes the cloning, expression, and purification of recombinant Nsa1 for structural determination by X-ray crystallography and small-angle X-ray scattering (SAXS), and is applicable for the hybrid structural analysis of other proteins containing both ordered and disordered domains.

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Immunology and Infection

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture
Andrea Introini 1,2, Christophe Vanpouille 2, Wendy Fitzgerald 2, Kristina Broliden 1, Leonid Margolis 2
1Department of Medicine Solna, Center for Molecular Medicine, Karolinska University Hospital, Karolinska Institutet, 2Section of Intercellular Interactions, Eunice Shriver National Institute of Child Health and Human Development, National Institutes of Health

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.

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Bioengineering

Probing the Roles of Physical Forces in Early Chick Embryonic Morphogenesis
Yan Li *1, Hannah Grover *1, Eric Dai 2, Kevin Yang 1, Zi Chen 1
1Thayer School of Engineering, Dartmouth College, 2Department of Bioengineering, University of Pennsylvania

Here, we present a protocol introducing a set of new ex-ovo experiments and physical modeling approaches for studying the mechanics of morphogenesis during early chick embryonic brain torsion.

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JoVE Journal

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies
Xuesong Feng *1, Faiza Naz *2, Aster H. Juan 1, Stefania Dell'Orso 2, Vittorio Sartorelli 1
1Laboratory of Muscle Stem Cells and Gene Regulation, National Institutes of Health, 2Office of Science and Technology, National Institute of Arthritis, Musculoskeletal and Skin Diseases (NIAMS), National Institutes of Health

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

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Developmental Biology

Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution
Tomoko Yamazaki 1,2, Wenling Li 1, Yoh-Suke Mukouyama 1
1Laboratory of Stem Cell and Neuro-Vascular Biology, Genetics and Developmental Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, 2Earle A. Chiles Research Institute, Robert W. Franz Cancer Center, Providence Portland Medical Center

Here, we describe a high resolution whole-mount imaging method in the entire adult mouse ear skin, which enables us to visualize branching morphogenesis and patterning of peripheral nerves and blood vessels, as well as immune cell distribution.

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Behavior

Noninvasive, High-throughput Determination of Sleep Duration in Rodents
R. Michelle Saré 1, Abigail Lemons 1, Anita Torossian 1, Carolyn Beebe Smith 1
1Section on Neuroadaptation and Protein Metabolism, National Institute of Mental Health, National Institutes of Health

We describe a high-throughput method of measuring sleep by means of activity-based home-cage monitoring. This method offers advantages over traditional EEG-based methods. It is well validated for the determination of total sleep duration and can be a powerful tool to monitor sleep in rodent models of human disease.

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Biology

Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis
Caleb Sutherland *1, Yu Wang *2, Robert V. Brown *1, Julie Foley 2, Beth Mahler 2, Kyathanahalli S. Janardhan 2,3, Ramesh C. Kovi 2,4, Anton M. Jetten 1
1Immunity, Inflammation, and Disease Laboratory, Division of Intramural Research, National Institute of Environmental Health Sciences, NIH, 2Cellular and Molecular Pathology Branch, Division of the National Toxicology Program, National Institute of Environmental Health Sciences, NIH, 3Integrated Laboratory Systems Inc., 4Experimental Pathology Laboratories Inc.

Here, we describe a protocol for a reproducible laser capture microdissection (LCM) for isolating trabecular meshwork (TM) for downstream RNA analysis. The ability to analyze changes in gene expression in the TM will help in understanding the underlying molecular mechanisms of TM-related ocular diseases.

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Neuroscience

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains
Giulia Quattrocolo 1, Maria Isaac 2, Yajun Zhang 2, Timothy J. Petros 2
1Kavli Institute for Systems Neuroscience and Centre for Neural Computation, Norwegian University of Science and Technology, 2Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health

Challenging young neurons in new brain regions can reveal important insights into how the environment sculpts neuronal fate and maturation. This protocol describes a procedure to harvest interneuron precursors from specific brain regions and transplant them either homotopically or heterotopically into the brain of postnatal pups.

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Cancer Research

Evaluating the Role of Mitochondrial Function in Cancer-related Fatigue
Li Rebekah Feng 1, Quang Nguyen 1, Alexander Ross 1, Leorey N. Saligan 1
1National Institute of Nursing Research, National Institutes of Health

Our goal was to develop a practical protocol to evaluate mitochondrial dysfunction associated with fatigue in cancer patients. This innovative protocol is optimized for clinical use involving only standard phlebotomy and basic laboratory procedures.

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Behavior

Chronic Sleep Deprivation in Mouse Pups by Means of Gentle Handling
Abigail Lemons 1, R. Michelle Saré 1, Carolyn Beebe Smith 1
1Section on Neuroadaptation and Protein Metabolism, National Institute of Mental Health, National Institutes of Health

We describe a sleep deprivation technique known as gentle handling where investigators gently prod mice any time sleep behavior is observed. This method is a powerful tool that allows researchers to study the effects that chronic sleep restriction throughout development can have on future brain physiology and behavior.

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JoVE Journal

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction
Dan Zhou *1,2, Lei Tan *1, Jian Li *3, Tanbin Liu 1, Yi Hu 1, Yalan Li 1, Sachiyo Kawamoto 4, Chengyu Liu 5, Shiyin Guo 3, Aibing Wang 1
1Lab of Animal Models and Functional Genomics (LAMFG), The Key Laboratory of Animal Vaccine & Protein Engineering, College of Veterinary Medicine, Hunan Agricultural University (HUNAU), 2Department of Pathology, Georgetown University Medical School, 3College of Food Science and Technology, Hunan Agricultural University (HUNAU), 4Lab of Molecular Cardiology (LMC), National Heart, Lung, and Blood Institute (NHLBI)/National Institutes of Health (NIH), 5Transgenic Core, National Heart, Lung, and Blood Institute (NHLBI)/National Institutes of Health (NIH)

Here, we present a detailed protocol for identifying homologous recombination events that occurred in mouse embryonic stem cells using Southern blotting and/or PCR. This method is exemplified by the generation of nonmuscle myosin II genetic replacement mouse models using traditional embryonic stem cell-based homologous recombination-mediated targeting technology.

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JoVE Core

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT
Katherine Kim 1, Shan Huang 2, Laura A. Fletcher 1, Alana E. O'Mara 1, Ilan Tal 3, Robert J. Brychta 1, Aaron M. Cypess 1, Kong Y. Chen 1, Brooks P. Leitner 1
1Diabetes, Endocrinology, and Obesity Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 2National Cancer Institute, National Institutes of Health, 3Division of Nuclear Medicine and Molecular Imaging, Department of Radiology, Beth Israel Deaconess Medical Center, Harvard Medical School

Using free, open-source software, we have developed an analytical approach to quantify total and regional brown adipose tissue (BAT) volume and metabolic activity of BAT using 18F-FDG PET/CT.

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Neuroscience

Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo
R. Michelle Saré 1, Anita Torossian 1, Michael Rosenheck 1, Tianjian Huang 1, Carolyn Beebe Smith 1
1Section on Neuroadaptation and Protein Metabolism, National Institute of Mental Health, National Institutes of Health

Protein synthesis is a critical biological process for cells. In brain, it is required for adaptive changes. Measurement of rates of protein synthesis in the intact brain requires careful methodological considerations. Here we present the L-[1-14C]-leucine quantitative autoradiographic method for determination of regional rates of cerebral protein synthesis in vivo.

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Medicine

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation
Sirada Srihirun 1,2, Alan N. Schechter 2, Barbora Piknova 2
1Department of Pharmacology, Faculty of Dentistry, Mahidol University, 2Molecular Medicine Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health

Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

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Bioengineering

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts
Armel N. Femnou 1,2, Abigail Giles 1, Robert S. Balaban 1
1Laboratory of Cardiac Energetics, National Heart, Lung, and Blood Institute, National Institutes of Health, 2Department of Biomedical Engineering, The George Washington University

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

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Environment

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants
Zhuo Li 1,2, Fangting Ai 3, Jing Zhang 4, Zhenyang Yu 1,2, Daqiang Yin 1,2
1College of Environmental Sciences and Engineering, Tongji University, 2Shanghai Institute of Pollution Control and Ecological Security, 3Jiaxing Tongji Institute of Environment, 4College of Ecological Technique and Engineering, Shanghai Institute of Technology

Trans- and multi-generational effects of persistent chemicals are essential in judging their long-term consequences in the environment and on the human health. We provide novel detailed methods for studying trans- and multi-generational effects using free-living nematode Caenorhabditis elegans.

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Developmental Biology

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution
Leighton H. Duncan 1,5, Mark W. Moyle 1,5, Lin Shao 1,5, Titas Sengupta 1,5, Richard Ikegami 1,5, Abhishek Kumar 4,5, Min Guo 4,5, Ryan Christensen 4,5, Anthony Santella 2,5, Zhirong Bao 2,5, Hari Shroff 4,5, William Mohler 3,5, Daniel A. Colón-Ramos 1,5,6
1Department of Neuroscience and Department of Cell Biology, Yale University School of Medicine, 2Developmental Biology Program, Sloan Kettering Institute, 3Department of Genetics and Genome Sciences and Center for Cell Analysis and Modeling, University of Connecticut Health Center, 4Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, 5WormGUIDES.org, 6Instituto de Neurobiología, Recinto de Ciencias Médicas, Universidad de Puerto Rico

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

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Education

Home-Based Prescribed Pulmonary Exercise in Patients with Stable Chronic Obstructive Pulmonary Disease
Xiaodan Liu 1,2, Peijun Li 3, Jian Li 3, Lu Xiao 1, Ning Li 3, Yufan Lu 3, Zhengrong Wang 3, Jianqing Su 3, Zhenwei Wang 4, Chunlei Shan 1,2, Weibing Wu 3
1School of Rehabilitation Science, Shanghai University of Traditional Chinese Medicine, 2Institute of Rehabilitation Medicine, Shanghai Academy of Traditional Chinese Medicine, 3Department of Sports Medicine, Shanghai University of Sport, 4Department of Respiratory Medicine, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine

Presented here is a protocol to investigate the effects of home-based prescribed pulmonary exercise in stable chronic obstructive pulmonary disease (COPD) patients, which is modified based on traditional Chinese exercises according to dyspnea and limited exercise capacity observed in COPD patients.

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Medicine

A Cryoinjury Model to Study Myocardial Infarction in the Mouse
Dong Wang *1,2, Grigol Tediashvili *1,2, Xiaomeng Hu 1,2, Alessia Gravina 2, Sivan G. Marcus 1,2, Hao Zhang 4, Jeffrey E Olgin 4, Tobias Deuse 1,2,5, Sonja Schrepfer 1,2,3,5
1Transplant and Stem Cell Immunobiology Lab, University Heart Center, 2Department of Surgery, Transplant and Stem Cell Immunobiology Lab, University of California San Francisco, 3Cardiovascular Research Center (CVRC) and DZHK German Center for Cardiovascular Research, 4Division of Cardiology, Cardiovascular Research Institute, University of California San Francisco, 5Cardiovascular Surgery, University Heart Center

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

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JoVE Journal

Thermocapillary Convection Space Experiment on the SJ-10 Recoverable Satellite
Li Duan *1,2, Yongli Yin *3, Jia Wang *1, Qi Kang 1,2, Di Wu 1, Huan Jiang 1, Pu Zhang 1, Liang Hu 1
1National Microgravity Laboratory, Institute of Mechanics, Chinese Academy of Sciences, 2School of Engineering Sciences, University of Chinese Academy of Sciences, 3China Astronaut Research and Training Center

A protocol for the space payload design, the space experiment on thermocapillary convection, and analyses of experimental data and images are presented in this paper.

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Biochemistry

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors
Laszlo Radnai 1,2, Rebecca F. Stremel 1,2, James R. Sellers 3, Gavin Rumbaugh 2, Courtney A. Miller 1,2
1Department of Molecular Medicine, The Scripps Research Institute, 2Department of Neuroscience, The Scripps Research Institute, 3Laboratory of Molecular Physiology, NHLBI, National Institutes of Health

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 µL per well. The platform should be applicable to virtually any ADP producing enzyme.

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Immunology and Infection

In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure
Myles X. Hodge 1, Sky W. Reece 1, Jennifer H. Madenspacher 2, Kymberly M. Gowdy 1
1Department of Pharmacology and Toxicology, East Carolina University, 2Research Triangle Park, National Institute of Environmental Health and Sciences

This manuscript describes a protocol for determining whether exposure to ozone, a criteria air pollutant, impairs alveolar macrophage efferocytosis in vivo. This protocol utilizes commonly used reagents and techniques and can be adapted to multiple models of pulmonary injury to determine effects on alveolar macrophage efferocytosis.

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Neuroscience

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons
Chengyu Sheng 1, Uzma Javed 1, Justin Rosenthal 1, Jun Yin 1, Bo Qin 1, Quan Yuan 1
1National Institute of Neurological Disorders and Stroke, National Institutes of Health

Here, we describe the method we employed to image highly motile dendritic filopodia in a live preparation of the Drosophila larval brain, and the protocol we developed to quantify time-lapse 3D imaging datasets for quantitative assessments of dendrite dynamics in developing neurons.

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Neuroscience

An Implantable System For Chronic In Vivo Electromyography
David Zealear 1, Yike Li 1, Shan Huang 1
1Department of Otolaryngology, Vanderbilt University Medical Center

Presented here is a protocol for the manufacturing of an implantable system for in vivo chronological recording of evoked and spontaneous electromyographic potentials. The system is applied to the investigation of reinnervation of laryngeal muscles following nerve injury.   

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Developmental Biology

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development
Renat N. Khaliullin 1,2,3, Jeffrey M. Hendel 1,2, Adina Gerson-Gurwitz 1,2, Shaohe Wang 1,4,5, Stacy D. Ochoa 1,6, Zhiling Zhao 1,7, Arshad Desai 1,2, Karen Oegema 1,2, Rebecca A. Green 1,2
1Ludwig Institute for Cancer Research, San Diego, 2Department of Cellular and Molecular Medicine, University of California, San Diego, 3Recursion Pharmaceuticals, 4Biomedical Sciences Graduate Program, University of California, San Diego, 5Cell Biology Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, 6Department of Biology, San Diego State University, 7Developmental and Stem Cell Biology Graduate Program, University of California, San Francisco

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80–100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

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Immunology and Infection

Analysis of Group IV Viral SSHHPS Using In Vitro and In Silico Methods
Xin Hu 1, Jaimee R. Compton 2, Patricia M. Legler 2
1National Center for Advancing Translational Sciences, National Institutes of Health, 2United States Naval Research Laboratory

We present a general protocol for identifying short stretches of homologous host-pathogen protein sequences (SSHHPS) embedded in the viral polyprotein. SSHHPS are recognized by viral proteases and direct the targeted destruction of specific host proteins by several Group IV viruses.

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Biology

Quantification of Proliferative and Dead Cells in Enteroids
Hua-Shan Li *1, Shao-Fang Xu *1, Jian-Ying Sheng *1, Zhi-Hui Jiang 1, Jing Wang 1, Ning Ding 1, Tao Wang 1, Matthew A. Odenwald 2, Jerrold R. Turner 2,3, Wei-Qi He 1, Hong Xu 1, Juan-Min Zha 1
1Jiangsu Key Laboratory of Neuropsychiatric Diseases and Cambridge-Suda (CAM-SU) Genomic Resource Center, Medical College of Soochow University, Department of Oncology, The First Affiliated Hospital of Soochow University, 2Department of Pathology, University of Chicago, 3Department of Pathology, Brigham and Women's Hospital–Harvard Medical School

The presented protocol uses flow cytometry to quantify the number of proliferating and dead cells in cultured mouse enteroids. This method is helpful to evaluate the effects of drug treatment on organoid proliferation and survival.

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Developmental Biology

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells
Kilsoo Jeon 1, Kyeyoon Park 2, Anton M. Jetten 1
1Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, 2NIH Stem Cell Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

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Biology

Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization
Xueshu Xie 1, Samah Shah 1, Anja Holtz 1, Jacob Rose 1, Nathan Basisty 1, Birgit Schilling 1
1Buck Institute for Research on Aging

This workflow describes the performance of time- and cost-efficient enrichment of multiple protein post-translational modifications (PTMs) simultaneously for quantitative global proteomic analysis. The protocol utilizes peptide-level PTM enrichment with multiple conjugated antibodies, followed by data-independent acquisition mass spectrometry analysis to gain biological insights into PTM crosstalk.

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Cancer Research

Measuring the Motor Aspect of Cancer-Related Fatigue using a Handheld Dynamometer
Li Rebekah Feng 1, Jeniece Regan 1, Joseph Shrader 2, Josephine Liwang 1, Sarah Alshawi 1, Jamell Joseph 2, Alexander Ross 1, Leorey Saligan 1
1National Institute of Nursing Research, National Institutes of Health, 2Clinical Center Rehabilitation Medicine, National Institutes of Health

Simple and accessible methods were developed to measure the motor aspect of cancer-related fatigue objectively and quantitatively. We describe, in detail, ways to administer the physical fatigue test using a simple handgrip device as well as methods to calculate fatigue indices.

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Neuroscience

Purification of Fibroblasts and Schwann Cells from Sensory and Motor Nerves in Vitro
Qianru He 1, Fanhui Yu 1, Yan Li 1, Junjie Sun 1, Fei Ding 1
1Key Laboratory of Neuroregeneration of Jiangsu and Ministry of Education, Co-Innovation Center of Neuroregeneration, Jiangsu Clinical Medicine Center of Tissue Engineering and Nerve Injury Repair, Nantong University

Here, we present a method to purify fibroblasts and Schwann cells from sensory and motor nerves in vitro.

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Engineering

Measurement of Chladni Mode Shapes with an Optical Lever Method
Rong Feng *1, Yan Luo *1, Yixue Dong 1, Mengke Ma 2, Yuqi Wang 2, Jing Zhang 3, Wenjiang Ma 3, Donghuan Liu 4
1School of Materials Science and Engineering, University of Science and Technology Beijing, 2School of Civil and Resource Engineering, University of Science and Technology Beijing, 3Basic Experimental Center for Natural Science, University of Science and Technology Beijing, 4School of Mathematics and Physics, University of Science and Technology Beijing

A simple method of measuring the Chladni mode shape on an elastic plate by the principle of an optical lever is proposed.

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Developmental Biology

Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging
Russell H. Knutsen 1, Leah M. Gober 1, Joseph R. Sukinik 1, Danielle R. Donahue 2, Elise K. Kronquist 1, Mark D. Levin 1, Sean E. McLean 3, Beth A. Kozel 1
1Translational Vascular Medicine Branch, National Heart Lung and Blood Institute, National Institutes of Health, 2Mouse Imaging Facility, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 3Division of Pediatric Surgery, Department of Surgery, University of North Carolina at Chapel Hill

The aim of this technique is ex vivo visualization of pulmonary arterial networks of early postnatal and adult mice through lung inflation and injection of a radio-opaque polymer-based compound via the pulmonary artery. Potential applications for casted tissues are also discussed.

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Biochemistry

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates
Monica C. Pillon 1, Robin E. Stanley 1
1Signal Transduction Laboratory, National Institute of Environmental Health Sciences, Department of Health and Human Services, National Institutes of Health

This protocol describes a nonradioactive assay to measure kinase activity of polynucleotide kinases (PNKs) on small DNA and RNA substrates.

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Neuroscience

Cerebrovascular Reactivity Measurement with Functional Near Infrared Spectroscopy
Franck Amyot 1, Cora Davis 1, Mike Sangobowale 4, Carol Moore 2, Erika Silverman 4, Amir Gandjbakhche 3, Ramon Diaz-Arrastia 4, Kimbra Kenney 1,2
1National Intrepid Center of Excellence, Walter Reed National Military Medical Center, 2Uniformed Services University of the Health Sciences, 3The Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, 4University of Pennsylvania Perelman School of Medicine

Presented here is a protocol for imaging and measurement of cerebrovascular reactivity in humans with functional Near Infrared Spectroscopy (fNIRS). fNIRS is a novel imaging modality that captures the concentration changes of hemoglobin species in the brain’s outermost cortex under specific stimuli.

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Immunology and Infection

Analysis of Human Natural Killer Cell Metabolism
Javier Traba 1,2, Thomas A. Waldmann 3, Olga M. Anton 3
1Cardiovascular Branch, National Heart, Lung and Blood Institute, National Institutes of Health, 2Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid (CSIC-UAM), 3Lymphoid Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health

In this paper, we describe a method to measure glycolysis and mitochondrial respiration in primary human Natural Killer (NK) cells isolated from peripheral blood, at rest or following IL15-induced activation. The protocol described could be easily extended to primary human NK cells activated by other cytokines or soluble stimuli.

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Chemistry

Direct-Coupled Electroretinogram (DC-ERG) for Recording the Light-Evoked Electrical Responses of the Mouse Retinal Pigment Epithelium
Kiyoharu J. Miyagishima 1, Congxiao Zhang *2, Volha V. Malechka *3, Kapil Bharti 2, Wei Li 1
1Retinal Neurophysiology Section, National Eye Institute, National Institutes of Health, 2Ocular and Stem Cell Translational Research Section, National Eye Institute, National Institutes of Health, 3Human Visual Function Core, National Eye Institute, National Institutes of Health

Here, we present a method for recording light-evoked electrical responses of the retinal pigment epithelium (RPE) in mice using a technique known as DC-ERGs first described by Marmorstein, Peachey, and colleagues in the early 2000s.

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Biochemistry

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology
Sayuri L. Higashi 1,2, Kazuya Yagyu 1,2, Haruna Nagase 1,2, Craig S. Pearson 1, Herbert M. Geller 1, Yasuhiro Katagiri 1
1Laboratory of Developmental Neurobiology, Cell and Developmental Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, 2United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University

An ultra-high-speed western blotting technique is developed by improving the kinetics of antigen-antibody binding through cyclic draining and replenishing (CDR) technology in conjunction with an immunoreaction enhancing agent.

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Biology

Visualization of Replisome Encounters with an Antigen Tagged Blocking Lesion
Jing Zhang *1, Jing Huang *2, Ishani Majumdar 1, Ryan C. James 3, Julia Gichimu 1, Manikandan Paramasivam 4, Durga Pokharel 5, Himabindu Gali 6, Marina A. Bellani 1, Michael M Seidman 1
1Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, 2Institute of Chemical Biology and Nanomedicine, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, Hunan University, 3Department of Molecular Biology and Genetics, Cornell University, 4Department of Cellular and Molecular Medicine, University of Copenhagen, 5Horizon Discovery, 6Boston University School of Medicine

While replication fork collisions with DNA adducts can induce double strand breaks, less is known about the interaction between replisomes and blocking lesions. We have employed the proximity ligation assay to visualize these encounters and to characterize the consequences for replisome composition.

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Bioengineering

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues
Ravi Lokwani 1, Kaitlyn Sadtler 1
1Section on Immuno-Engineering, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.

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JoVE Journal

Rapid Determination of Antibody-Antigen Affinity by Mass Photometry
Di Wu 1, Grzegorz Piszczek 1
1Biophysics Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health

We describe a single-molecule approach to antigen-antibody affinity measurements using mass photometry (MP). The MP-based protocol is fast, accurate, uses a very small amount of material, and does not require protein modification.

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Behavior

An Assessment Method and Toolkit to Evaluate Keyboard Design on Smartphones
Yincheng Wang 1, Ke Wang 1, Yuqi Huang 1, Di Wu 2, Jian Wu 3, Jibo He 1
1Department of Psychology, School of Social Sciences, Tsinghua University, 2Department of Computer Science, Beijing Normal University, 3Haier Innovation Design Center, Haier Company

The presented protocol integrates various evaluation methods and demonstrates a method to evaluate the keyboard design on smartphones. Pairs matched by English characters are proposed as the input material, and the transition time between two keys is used as the dependent variable.

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Cancer Research

Studying TGF-β Signaling and TGF-β-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells
Jing Zhang 1, Midory Thorikay 1, Gerard van der Zon 1, Maarten van Dinther 1, Peter ten Dijke 1
1Oncode Institute and Department of Cell Chemical Biology, Leiden University Medical Center

We describe a systematic workflow to investigate TGF-β signaling and TGF-β-induced EMT by studying the protein and gene expression involved in this signaling pathway. The methods include Western blotting, a luciferase reporter assay, qPCR, and immunofluorescence staining.

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Engineering

Design and Development of a Three-Dimensionally Printed Microscope Mask Alignment Adapter for the Fabrication of Multilayer Microfluidic Devices
Celine R. Garcia *1, Zhenya Ding *1, Hilario C. Garza 1, Wei Li 1
1Department of Chemical Engineering, Texas Tech University

This project allows small laboratories to develop an easy-to-use platform for the fabrication of precise multilayer microfluidic devices. The platform consists of a three-dimensionally printed microscope mask alignment adapter using which multilayer microfluidic devices with alignment errors of <10 µm were achieved.

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Genetics

Sand Fly (Phlebotomus papatasi) Embryo Microinjection for CRISPR/Cas9 Mutagenesis
Isabelle Louradour 1, Kashinath Ghosh 1, Ehud Inbar 1, David L. Sacks 1, Channa Aluvihare 2, Robert A. Harrell II 2
1Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 2University of Maryland Insect Transformation Facility, The Institute for Bioscience and Biotechnology Research

This protocol details the steps of CRISPR/Cas9 targeted mutagenesis in sand flies: embryo collection, injection, insect rearing, and identification as well as selection of mutations of interest.

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Genetics

R-Loop Analysis by Dot-Blot
Prisila Ramirez 1, Robert J. Crouch 2, Vivian G. Cheung 1,3,4, Christopher Grunseich 5
1Life Sciences Institute, University of Michigan, Ann Arbor, 2Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, 3Department of Pediatrics, University of Michigan, Ann Arbor, 4Howard Hughes Medical Institute, 5Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health

This protocol details a simple method that quantifies R-loop, a three-stranded nucleic acid structure that comprises of an RNA-DNA hybrid and a displaced DNA strand.

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Biochemistry

Transplantation of Neonatal Mouse Cardiac Macrophages into Adult Mice
Yandong Li 1, Jie Feng 1, Yan Li 1, Jianqiu Pei 1, Shengshou Hu 1, Yu Nie 1,2
1Fuwai Hospital & Chinese Academy of Medical Sciences, State Key Laboratory of Cardiovascular Disease, Cardiovascular Institute, National Center for Cardiovascular Diseases, Peking Union Medical College, 2National Health Commission Key Laboratory of Cardiovascular Regenerative Medicine, Fuwai Central-China Hospital, Central-China Subcenter of National Center for Cardiovascular Diseases

We provide a protocol for neonatal cardiac macrophage separation and transplantation into an adult mouse heart, which could be a promising way to promote cardiac repair.

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JoVE Journal

Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays
Ananya Tripathi 1, Charles Bond 1,2, James R. Sellers 1, Neil Billington 1, Yasuharu Takagi 1
1Laboratory of Molecular Physiology, Cell and Developmental Biology Center, National Heart, Lung and Blood Institute, National Institutes of Health, 2Department of Physiology, Perelman School of Medicine, University of Pennsylvania

Presented here is a procedure to express and purify myosin 5a followed by a discussion of its characterization, using both ensemble and single molecule in vitro fluorescence microscopy-based assays, and how these methods can be modified for the characterization of nonmuscle myosin 2b.

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Biology

Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements
Ji Won Park *1, Samantha M. Thomas *1, Lee J. Wylie 2, Andrew M. Jones 2, Anni Vanhatalo 2, Alan N. Schechter 1, Barbora Piknova 1
1Molecular Medicine Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 2Sport and Health Sciences, College of Life and Environmental Sciences, St Luke’s Campus, University of Exeter

We present protocols for three different methods for the homogenization of four different muscle groups of rat skeletal muscle tissue to measure and compare the levels of nitrate and nitrite. Furthermore, we compare different sample weights to investigate whether tissue sample size affects the results of homogenization.

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Neuroscience

Acquisition of Resting-State Functional Magnetic Resonance Imaging Data in the Rat
Diana J. Wallin *1,2, Emily D. K. Sullivan *1,2, Elise M. Bragg 1, Jibran Y. Khokhar 2,4, Hanbing Lu 2,3, Wilder T. Doucette 1,2
1Dartmouth-Hitchcock Medical Center, 2Geisel School of Medicine at Dartmouth, 3National Institute on Drug Abuse, National Institutes of Health, 4University of Guelph

This protocol describes a method for obtaining stable resting-state functional magnetic resonance imaging (rs-fMRI) data from a rat using low dose isoflurane in combination with low dose dexmedetomidine.

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Biochemistry

Polysome Profiling without Gradient Makers or Fractionation Systems
Mack Sobhany 1, Robin E. Stanley 1
1Signal Transduction Laboratory, National Institute of Environmental Health Sciences, Department of Health and Human Services, National Institutes of Health

This protocol describes how to generate a polysome profile without using automated gradient makers or gradient fractionation systems.

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Neuroscience

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples
Ke Jiang 1, Jacob Nellissery 1, Anand Swaroop 1
1Neurobiology, Neurodegeneration & Repair Laboratory, National Eye Institute, National Institutes of Health

Described here is a detailed protocol for performing mitochondrial stress assay and glycolytic rate assay in ex vivo retinal tissue samples using a commercial bioanalyzer.

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Genetics

Reusable Single Cell for Iterative Epigenomic Analyses
Hidetaka Ohnuki 1, David J. Venzon 2, Alexei Lobanov 3,4, Giovanna Tosato 1
1Laboratory of Cellular Oncology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 2Biostatistics and Data Management Section, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 3CCR Collaborative Bioinformatics Resource (CCBR), Center for Cancer Research, National Cancer Institute, National Institutes of Health, 4Advanced Biomedical Computational Science, Frederick National Laboratory for Cancer Research sponsored by the National Cancer Institute

The present protocol describes a single-cell method for iterative epigenomic analyses using a reusable single cell. The reusable single cell allows analyses of multiple epigenetic marks in the same single cell and statistical validation of the results.

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Neuroscience

Intraventricular Drug Delivery and Sampling for Pharmacokinetics and Pharmacodynamics Study
Sara Oberrauch 1, Jing Lu 1, Linda Cornthwaite-Duncan 1, Maytham Hussein 1, Jian Li 2, Gauri Rao 3, Tony Velkov 1
1Department of Biochemistry & Pharmacology, School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, 2Department of Microbiology, Biomedicine Discovery Institute, Monash University, 3UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill

Delivery of therapeutics directly into the central nervous system is one way of circumventing the blood-brain barrier. The present protocol demonstrates intracerebroventricular injection for subsequent collection of cerebrospinal fluid and bodily organs. This facilitates the investigation of drug pharmacokinetics and pharmacodynamics in animal models for developing new treatments.

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Biochemistry

Purification of Ubiquitinated p53 Proteins from Mammalian Cells
Di Wu 1, Mengfan He 1, Dawei Li 1
1Center for Translational Medicine, The Affiliated Zhangjiagang Hospital of Soochow University

The protocol describes a step-by-step method to purify ubiquitinated proteins from mammalian cells using the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified from cells under stringent nondenaturing and denaturing conditions.

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Cancer Research

Digital Spatial Profiling for Characterization of the Microenvironment in Adult-Type Diffusely Infiltrating Glioma
Nishika Karbhari 1, Rachael Barney 2, Scott Palisoul 2, Jennifer Hong 3, Chun-Chieh Lin 2, George Zanazzi 2,4
1Department of Neurology, Dartmouth-Hitchcock Medical Center, 2Department of Pathology and Laboratory Medicine, Dartmouth-Hitchcock Medical Center, 3Department of Neurosurgery, Dartmouth-Hitchcock Medical Center, 4Dartmouth Cancer Center, Dartmouth-Hitchcock Medical Center

Proteomic dysregulation plays an important role in the spread of diffusely infiltrating gliomas, but several relevant proteins remain unidentified. Digital spatial processing (DSP) offers an efficient, high-throughput approach for characterizing the differential expression of candidate proteins that may contribute to the invasion and migration of infiltrative gliomas.

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Biology

Intravital Subcellular Microscopy of the Mammary Gland
Yeap Ng 1, Andrius Masedunskas 1,2, Marco Heydecker 1, Seham Ebrahim 1,3, Roberto Weigert 1, Ian H. Mather 1
1Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 2Charles Perkins Centre, Central Clinical School, Faculty of Medicine and Health, The University of Sydney, 3Department of Molecular Physiology and Biological Physics, School of Medicine, University of Virginia

The present protocol describes a facile technique for the intravital imaging of the lactating mouse mammary gland by laser scanning confocal and multiphoton microscopy.

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Biochemistry

Measurement of Protein Turnover Rates in Senescent and Non-Dividing Cultured Cells with Metabolic Labeling and Mass Spectrometry
Matthew Payea 1, Myriam Gorospe 1, Nathan Basisty 2
1Laboratory of Genetics and Genomics, National Institute on Aging Intramural Research Program, National Institutes of Health, 2Translational Gerontology Branch, National Institute on Aging Intramural Research Program, National Institutes of Health

This protocol describes the workflow for metabolic labeling of senescent and non-dividing cells with pulsed SILAC, untargeted mass spectrometry analysis, and a streamlined calculation of protein half-lives.

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Biology

Sample Preparation for Rapid Lipid Analysis in Drosophila Brain Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging
Yuki X. Chen *1,5,6, Kelly Veerasammy *1,2, Jun Yin *3, Tenzin Choetso 1,4, Tiffany Zhong 7, Muniyat A. Choudhury 1,4,5, Cory Weng 1,4,5, Ethan Xu 8, Mayan A. Hein 9,10, Rinat Abzalimov 2, Ye He 1
1Advanced Science Research Center, Neuroscience Initiative, the City University of New York, Graduate Center New York, 2Advanced Science Research Center, Structural Biology Initiative, the City University of New York, Graduate Center, 3National Institute of Neurological Disorders and Stroke, National Institutes of Health, 4The City College of New York, CUNY, 5Macaulay Honors College, CUNY, 6Graduate School of Public Health and Health Policy, The City University of New York, 7Princeton University, 8Ardrey Kell High School, 9The Borough of Manhattan Community College, CUNY, 10Gallatin School of Individualized Study, New York University

The aim of this protocol is to provide detailed guidance on the proper sample preparation for lipid and metabolite analysis in small tissues, such as the Drosophila brain, using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging.

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Developmental Biology

FACS-Isolation and Culture of Fibro-Adipogenic Progenitors and Muscle Stem Cells from Unperturbed and Injured Mouse Skeletal Muscle
Giulia Riparini 1, James M. Simone 2, Vittorio Sartorelli 1
1Laboratory of Muscle Stem Cells and Gene Regulation, National Institute of Arthritis, Musculoskeletal, and Skin Diseases (NIAMS), National Institutes of Health (NIH), 2Flow Cytometry Section, National Institute of Arthritis, Musculoskeletal, and Skin Diseases (NIAMS), National Institutes of Health (NIH)

The precise identification of fibro-adipogenic progenitor cells (FAPs) and muscle stem cells (MuSCs) is critical to studying their biological function in physiological and pathological conditions. This protocol provides guidelines for the isolation, purification, and culture of FAPs and MuSCs from adult mouse muscles.

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Environment

Visualizing Field Data Collection Procedures of Exposure and Biomarker Assessments for the Household Air Pollution Intervention Network Trial in India
Karthikeyan D. Rajamani 1, Sankar Sambandam 1, Krishnendu Mukhopadhyay 1, Naveen Puttaswamy 1, Gurusamy Thangavel 1, Durairaj Natesan 1, Rengaraj Ramasamy 1, Saritha Sendhil 1, Amudha Natarajan 1, Vigneswari Aravindalochan 1, Ajay Pillarisetti 2, Michael Johnson 3, Joshua Rosenthal *4, Kyle Steenland 5, Ricardo Piedhrahita 3, Jennifer Peel 6, Maggie L. Clark 6, Dana Boyd Barr 5, Sarah Rajkumar 6, Bonnie Young 6, Shirin Jabbarzadeh 7, Ghislaine Rosa 8, Miles Kirby 9, Lindsay J. Underhill 10, Anaite Diaz-Artiga 11, Amy Lovvorn 5, William Checkley 12, Thomas Clasen 5, Kalpana Balakrishnan 1
1Department of Environmental Health Engineering, ICMR Center for Advanced Research on Air Quality, Climate and Health, Faculty of Public Health, Sri Ramachandra Institute of Higher Education and Research (Deemed University), 2Division of Environmental Health Sciences, University of California, Berkeley, 3Berkeley Air Monitoring Group, 4Division of International Epidemiology and Population Studies, National Institutes of Health, 5Gangarosa Department of Environmental Health, Rollins School of Public Health, Emory University, 6Department of Environmental and Radiological Health Sciences, Colorado State University, 7Department of Biostatistics and Informatics, Rollins School of Public Health, Emory University, 8Department of Disease Control, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, 9Department of Global Health & Population, Harvard, T.H. Chan School of Public Health, 10Cardiovascular Division, Washington University School of Medicine, Washington University, 11Centro de Estudios en Salud, Universidad del Valle de Guatemala, 12Division of Pulmonary and Critical Care, School of Medicine, Johns Hopkins University

We detail the consistent, high-quality procedures used throughout air and biological sampling processes at Indian field sites during a large randomized controlled trial. Insights gathered from the oversight of applications of innovative technologies, adapted for exposure assessment in rural regions, enable better field data collection practices with more reliable outcomes.

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Biology

Primary Culture of Porcine Retinal Pigment Epithelial Cells
Feng Wen *1,2,3, Yanzi Wang *1,2,3, Danxue He 1,2,3, Chunyan Liao 1,2,3, Weijie Ouyang 1,2,3, Zuguo Liu 1,2,3, Wei Li 1,2,3, Yi Liao 1,2,3
1Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, School of Medicine, Xiamen University, 2Department of Ophthalmology, Xiang'an Hospital of Xiamen University, School of Medicine, Xiamen University, 3Xiamen University Affiliated Xiamen Eye Center, School of Medicine, Xiamen University

Here, an easy-to-follow method to culture primary porcine retinal pigment epithelial cells in vitro is presented.

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Cancer Research

Co-Culture and Transduction of Murine Thymocytes on Delta-Like 4-Expressing Stromal Cells to Study Oncogenes in T-Cell Leukemia
Gisele O. L. Rodrigues 1, WenQing Li 1, Sarah D. Cramer 1,2,3, Hila Y. Winer 1, Tu Chun Hsu 1,2,3, Timothy Gower 4, Julie A. Hixon 1, Scott K. Durum 1
1Cytokines and Immunity Section, Cancer Innovation Laboratory, National Cancer Institute, National Institutes of Health, 2Comparative Biomedical Scientist Training Program, NIH, 3Department of Pathobiology and Diagnostic Investigation, Veterinary Diagnostic Laboratory, Michigan State University, 4NCI-Frederick Laboratory Animal Sciences Program

This protocol describes the isolation of double-negative thymocytes from the mouse thymus followed by retroviral transduction and co-culture on the delta-like 4-expressing bone marrow stromal cell line co-culture system (OP9-DL4) for further functional analysis.

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Medicine

Rodent Model of Intestinal Ischemia-Reperfusion Injury via Occlusion of the Superior Mesenteric Artery
Lucie Henein 1,2, Randy Clevenger 1, Karen Keeran 1, Lauren Brinster 3
1Animal Surgery and Resources Core, National Heart, Lung, and Blood Institute, National Institutes of Health, 2College of Veterinary Medicine, Mississippi State University, 3Division of Veterinary Resources, Office of Research Services, National Institutes of Health

We describe how to generate a widely used surgical model of intestinal ischemia-reperfusion injury (IRI) in rodents. The procedure involves occlusion of the superior mesenteric artery followed by the restoration of blood flow. This model is useful for studies investigating occlusive causes of intestinal IRI in both veterinary and human medicine.

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Immunology and Infection

Rat Burn Model to Study Full-Thickness Cutaneous Thermal Burn and Infection
Rajnikant Sharma 1, Shekhar Yeshwante 1, Quentin Vallé 1, Maytham Hussein 2, Varsha Thombare 2, Sean Michael McCann 1, Robert Maile 3,4,5, Jian Li 6, Tony Velkov 2, Gauri Rao 1
1UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, 2Department of Biochemistry & Pharmacology, School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, 3Department of Microbiology & Immunology, University of North Carolina School of Medicine, 4Department of Surgery, University of North Carolina at Chapel Hill, 5Curriculum in Toxicology and Environmental Medicine, University of North Carolina at Chapel Hill, 6Department of Microbiology, Monash Biomedicine Discovery Institute, Monash University

A model mimicking the clinical scenario of burn injury and infection is necessary for furthering burn research. The present protocol demonstrates a simple and reproducible rat burn infection model comparable to that in humans. This facilitates the study of burn and infections following burn for developing new topical antibiotic treatments.

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Medicine

Establishing a Silicosis Rat Model via Exposure of Whole-Body to Respirable Silica
Fuyu Jin 1, Yaqian Li 1, Tian Li 1, Xinyu Yang 1, Wenchen Cai 1, Shifeng Li 1, Xuemin Gao 1, Fang Yang 1, Hong Xu 1, Heliang Liu 1
1Hebei Key Laboratory for Organ Fibrosis Research, School of Public Health, North China University of Science and Technology

This study describes a technique to establish a silicosis rat model with the inhalation of silica through the whole body in an inhalation chamber. The rats with silicosis could closely mimic the pathological process of human silicosis in an easy, cost-effective manner with good repeatability.

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Biology

Efficient and Consistent Generation of Retinal Pigment Epithelium/Choroid Flatmounts from Human Eyes for Histological Analysis
Davide Ortolan 1, Andrei Volkov 1, Arvydas Maminishkis 1, Ruchi Sharma 1, Kapil Bharti 1
1Ocular and Stem Cell Translational Research Section, National Eye Institute, National Institutes of Health (NIH)

We describe a method to efficiently separate retinal pigment epithelium (RPE) from the retina in human eyes and generate whole RPE/choroid flatmounts for histological and morphometric analyses of the RPE.

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Immunology and Infection

Gene Editing of Primary Rhesus Macaque B Cells
Harald Hartweger 1, Rajeev Gautam 2, Yoshiaki Nishimura 2, Fabian Schmidt 3,5, Kai-Hui Yao 1, Amelia Escolano 1,6, Mila Jankovic 1, Malcolm A. Martin 2, Michel C. Nussenzweig 1,4
1Laboratory of Molecular Immunology, The Rockefeller University, 2Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 3Laboratory of Retrovirology, The Rockefeller University, 4Howard Hughes Medical Institute, The Rockefeller University, 5Laboratory of Applied Virology and Precision Medicine, King Abdullah University of Science and Technology (KAUST), 6Vaccine and Immunotherapy Center, Wistar Institute

We present a method for culturing and gene editing primary rhesus macaque B cells using CRISPR/Cas9 and recombinant adeno-associated virus serotype 6 for the study of B cell therapies.

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Medicine

Ferric Chloride-Induced Arterial Thrombosis and Sample Collection for 3D Electron Microscopy Analysis
Smita Joshi *1, Alexis N. Smith 1, Kanakanagavalli Shravani Prakhya 1, Hammodah R. Alfar 1, Joshua Lykins 1, Ming Zhang 1, Irina Pokrovskaya 2, Maria Aronova 3, Richard D. Leapman 3, Brian Storrie 2, Sidney W. Whiteheart *1
1Department of Molecular and Cellular Biochemistry, University of Kentucky, 2Department of Physiology and Cell Biology, University of Arkansas for Medical Sciences, 3Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health

The present protocol describes how to use a FeCl3-mediated injury to induce arterial thrombosis, and how to collect and prepare arterial injury samples at various stages of thrombosis for electron microscopy analysis.

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Transgenic Manipulation of Arthropod Vectors: Tools to Study Vector-Borne Diseases
Adeline E. Williams 1,2, Kenneth E. Olson 1
1Department of Microbiology, Immunology, Pathology, Colorado State University, 2Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health

Transgenic Manipulation of Arthropod Vectors: Tools to Study Vector-Borne Diseases

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Biology

Assessment of Chemical Toxicity in Adult Drosophila Melanogaster
Jessica M. Holsopple *1,2, Shannon R. Smoot *1, Ellen M. Popodi 1,2, John K. Colbourne 3, Joseph R. Shaw 4, Brian Oliver 5, Thomas C. Kaufman 1, Jason M. Tennessen 1
1Department of Biology, Indiana University, 2Bloomington Drosophila Stock Center, Department of Biology, Indiana University, 3School of Biosciences, University of Birmingham, 4O'Neill School of Public and Environmental Affairs, Indiana University, 5Section of Developmental Genomics, Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Kidney and Digestive Diseases, National Institutes of Health

This protocol describes an efficient and inexpensive method that uses liquid media to assess the effects of chemical toxicants on the viability of adult Drosophila melanogaster.

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Biology

Quantifying Fitness Costs in Transgenic Aedes aegypti Mosquitoes
Adeline E. Williams 1,2, Irma Sanchez-Vargas 1, Lindsay E. Martin 2,3, Ines Martin-Martin 2,4, Susi Bennett 1, Ken E. Olson 1, Eric Calvo 2
1Center for Vector-borne Infectious Diseases, Department of Microbiology, Immunology, and Pathology, Colorado State University, 2Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 3Department of Biological Sciences, Vanderbilt University, 4National Center for Microbiology, Instituto de Salud Carlos III

The present protocol describes how to measure common life parameter data in Aedes aegypti mosquitoes, including fecundity, wing size, fertility, sex ratio, viability, development times, male contribution, and adult longevity. These measurements can be used to assess the fitness of transgenic mosquitoes.

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Bioengineering

Microbial Control and Monitoring Strategies for Cleanroom Environments and Cellular Therapies
Amanda D. East 1, James E. T. Gebo 1, Anna F. Lau 1
1Sterility Testing Service, Department of Laboratory Medicine, Clinical Center, National Institutes of Health

The protocol summarizes the best practices to minimize microbial bioburden in a cleanroom environment and includes strategies such as environmental monitoring, process monitoring, and product sterility testing. It is relevant for manufacturing and testing facilities that are required to meet current good tissue practice standards and current good manufacturing practice standards.

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Biology

Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining
Dillon Strepay 1, Rafal Olszewski 1, Ian Taukulis 1, J. Dixon Johns 1, Shoujun Gu 1, Michael Hoa 1
1Auditory Development and Restoration Program, National Institute on Deafness and Other Communication Disorders, National Institutes of Health

The stria vascularis is vital to the generation of endocochlear potential. Here, we present the dissection of the adult mouse stria vascularis for single-nucleus sequencing or immunostaining.

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Biochemistry

Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications
Yilun Sun 1
1Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health

The present protocol highlights a modified method to detect and quantify DNA-protein crosslinks (DPCs) and their post-translational modifications (PTMs), including ubiquitylation, SUMOylation, and ADP-ribosylation induced by topoisomerase inhibitors and by formaldehyde, thereby allowing the study of the formation and repair of DPCs and their PTMs.

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Immunology and Infection

A Model for Experimental Exposure of Humans to Larval Ixodes scapularis Ticks
Siu Ping Turk 1, Aleah Eschman 1, Adriana Marques 1
1Lyme Disease Studies Unit, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy & Infectious Diseases, National Institutes of Health

This article presents the methodology for exposing humans to larval Ixodes scapularis for clinical research. The technique is relatively simple, tolerable by the research volunteers, and can be modified according to experimental needs. Such research involving human subjects must be conducted under clinical study protocols approved by the appropriate regulatory authorities.

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Dendritic Spine Quantification Using an Automatic Three-Dimensional Neuron Reconstruction Software

Dendritic Spine Quantification Using an Automatic Three-Dimensional Neuron Reconstruction Software
Kevin M. Keary III 1, Ellen Sojka 1,2, Melissa Gonzalez 1,2, Zheng Li 1
1Section on Synapse Development Plasticity, National Institute of Mental Health, National Institutes of Health, 2Colgate University

Dendritic spines are post-synaptic compartments of most excitatory synapses. Alterations to dendritic spine morphology occur during neurodevelopment, aging, learning, and many neurological and psychiatric disorders, underscoring the importance of reliable dendritic spine analysis. This protocol describes quantifying dendritic spine morphology accurately and reproducibly using automatic three-dimensional neuron reconstruction software.

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Genetics

Systemic Treatment for Postnatal, Juvenile, and Runted Adult Mice by Retrobulbar Sinus Injection
Darwin Romero 1, Randy J. Chandler 1
1Metabolic Medicine Branch, National Human Genome Institute, National Institutes of Health

This article provides a protocol and an accompanying video for the retrobulbar sinus injection of up to a total volume of 150 µL for postnatal, juvenile, and runted adult mice. This procedure is particularly well suited for the injection of small mice (15 g) when tail vein injection is not feasible.

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Medicine

Consistent Delivery of Adeno-Associated Virus via Lateral Tail-Vein Injection in Adult Mice
Fady Guirguis 1,2, Véronique Bolduc 1, Matthew J. Slarve 3, Haiyan Zhou 4,5, Francesco Muntoni 2,4, Carsten G. Bönnemann 1
1Neuromuscular and Neurogenetic Disorders of Childhood Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 2The Dubowitz Neuromuscular Centre, Molecular Neurosciences Section, Developmental Neurosciences Research and Teaching Department, Great Ormond Street Institute of Child Health, University College London, 3Department of Molecular Microbiology and Immunology, University of Southern California, 4NIHR Great Ormond Street Hospital Biomedical Research Centre, 5Genetics and Genomic Medicine Research and Teaching Department, Great Ormond Street Institute of Child Health, University College London

Here we detail an optimized protocol for mouse lateral tail-vein injection to systemically administer adeno-associated virus (AAV) in adult mice. Additionally, we describe protocols of commonly used assays to assess AAV transduction.

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Immunology and Infection

Preparation of Single-Cell Suspension of Mouse Thymic Epithelial Cells and Staining of Intracellular Molecules for Flow Cytometric Analysis
Mei-Ting Yang *1, Jie Li *1, Mami Matsuda-Lennikov *1, Assiatu Crossman 2, Yousuke Takahama 1
1Thymus Biology Section, Experimental Immunology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 2Flow Cytometry Facility, Experimental Immunology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health

We describe protocols for the preparation of single-cell suspension of mouse thymic epithelial cells and the staining of intracellular molecules for flow cytometric analysis.

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Biology

Understanding the Development of Compensatory Pathways in a Mutant Malaria Parasite Harbouring Hypomorphic Allele of Plant-Like Kinases
Himashree Choudhury *1, Manish Sharma *1, Utkarsh Gangwar 1, Nayla Siddiqui 1, Abhisheka Bansal 1,2
1Cellular and Molecular Parasitology Laboratory, School of Life Sciences, Jawaharlal Nehru University, 2Laboratory of Malaria and Vector Research and National Institutes of Allergy and Infectious Diseases, National Institutes of Health

Chemical genetics involves the substitution of a gatekeeper residue with an amino acid containing a different side chain at the target locus. Here, we have generated a mutant parasite containing a hypomorphic allele of cdpk1 and identified compensatory pathways adopted by the parasite in the mutant background.

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