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University of California, Davis

72 ARTICLES PUBLISHED IN JoVE

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Biology

Patterning Cells on Optically Transparent Indium Tin Oxide Electrodes
Sunny Shah 1, Alexander Revzin 1
1Department of Biomedical Engineering, University of California, Davis

Non-fouling PEG silane monolayer was desorbed from individually addressable ITO electrodes on glass by application of a reductive potential. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.

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Biology

Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture
Georgia Woods 1, Karen Zito 1
1Center for Neuroscience, University of California, Davis

We describe a method for preparing DNA coated gold bullets and demonstrate the use of such bullets to biolistically transfect neurons in cultured hippocampal slices.

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Biology

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus
Peter Armstrong 1,2, Mara Conrad 3
1Department of Molecular and Cell Biology, University of California, Davis, 2Marine Biological Laboratory - MBL- woods hole, 3Department of Biological Sciences, Hunter College of CUNY

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and α2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

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Biology

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo
Ingrid Brust-Mascher 1, Jonathan M. Scholey 1
1Department of Molecular and Cellular Biology, University of California, Davis

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

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Biology

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization
Bruce W. Draper 1, Cecilia B. Moens 2
1Molecular and Cellular Biology, University of California, Davis, 2Division of Basic Science, Fred Hutchinson Cancer Research Center - FHCRC

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

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Neuroscience

Mouse Models of Periventricular Leukomalacia
Yan Shen 1, Jennifer M. Plane 1, Wenbin Deng 1
1Department of Cell Biology and Human Anatomy Institute for Pediatric Regenerative Medicine School of, University of California, Davis

We established mouse models of periventricular leukomalacia (PVL), the predominant brain injury in premature infants characterized by periventricular white matter lesions. Hypoxia/ischemia with/without systemic infection are the primary causes of PVL. Unilateral carotid ligation and hypoxia exposure with/without lipopolysaccharide injection creates PVL-like lesions in P6 mice.

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Biology

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors
Peng Jiang 1, Vimal Selvaraj 1, Wenbin Deng 1
1Department of Cell Biology and Human Anatomy Institute for Pediatric Regenerative Medicine, School of Medicine, University of California, Davis

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

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Immunology and Infection

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy
Benjamin Dale 1, Gregory P. McNerney 2, Deanna L. Thompson 2, Wolfgang Hübner 3, Thomas Huser 2, Benjamin K. Chen 1
1Division of Infectious Diseases, Department of Medicine, Immunology Institute, Mount Sinai School of Medicine , 2NSF Center for Biophotonics, University of California, Davis, 3Structural and Computational Biology Unit, European Molecular Biology Laboratory

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

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Biology

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering (CARS)
Tyler J. Weeks 1, Thomas R. Huser 1,2
1Center for Biophotonics, University of California, Davis, 2Department of Internal Medicine, University of California, Davis

A combination of three single wavelength short-pulsed lasers is used to generate coherent anti-Stokes Raman scattering (CARS) and doubly-resonant CARS (DR-CARS). The difference between these signals provides enhanced sensitivity for otherwise difficult to detect coherent Raman signals, enabling imaging of weak Raman scatterers.

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Neuroscience

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila
Joanna C. Chiu 1,2, Kwang Huei Low 1,3, Douglas H. Pike 1, Evrim Yildirim 1,3, Isaac Edery 1,3
1Center for Advanced Biotechnology and Medicine, Rutgers University, 2Current Address: Department of Entomology, College of Agricultural and Environmental Sciences, University of California, Davis, 3Department of Molecular Biology and Biochemistry, Rutgers University

We describe procedures for recording daily locomotor activity rhythms of Drosophila and subsequent data analysis. Locomotor activity rhythms are a reliable behavioral output of animal circadian clocks and are used as the standard readout of clock function when studying circadian mutants or examining how the environment regulates the circadian system.

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Biology

Whole-Cell Recording of Calcium Release-Activated Calcium (CRAC) Currents in Human T Lymphocytes
Pratima Thakur 1, Alla F. Fomina 1
1Department of Physiology and Membrance Biology, University of California, Davis

We provide a step-by-step protocol for whole-cell patch clamp recording of Calcium Release-Activated Calcium (CRAC) currents in peripheral blood mononuclear cell-derived human T lymphocytes.

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Immunology and Infection

Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides
Nazzy Pakpour 1, Kong Wai Cheung 1, Lattha Souvannaseng 1, Jean-Paul Concordet 2, Shirley Luckhart 1
1Department of Medical Microbiology and Immunology, University of California, Davis, 2Département Génétique et Développement, Institut Cochin, Université Paris Descartes

Oligonucleotides can be used to site specifically substitute a single nucleotide of transfected target genes in both Anopheles gambiae and Anopheles stephensi cells.

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JoVE Core

Measurement of Bioelectric Current with a Vibrating Probe
Brian Reid 1, Min Zhao 1
1Dermatology, University of California, Davis

The manufacture, calibration and use of non-invasive vibrating probes to measure bioelectric current in various biological systems is described.

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Neuroscience

Electrophysiological Measurements from a Moth Olfactory System
Zainulabeuddin Syed 1, Walter S. Leal 1
1Department of Entomology, University of California, Davis

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively.

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Biology

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning
Elva Diaz 1, Gustavo A. Barisone 1
1Department of Pharmacology, University of California, Davis

We demonstrate the use of DNA microarrays for expression profiling of the nervous system. We describe RNA quality control, sample labeling, and array hybridization and scanning.

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Bioengineering

Rejection of Fluorescence Background in Resonance and Spontaneous Raman Microspectroscopy
Zachary J. Smith *1, Florian Knorr *1, Cynthia V. Pagba 1, Sebastian Wachsmann-Hogiu 1,2
1Center for Biophotonics Science and Technology, University of California, Davis, 2Department of Pathology and Laboratory Medicine, University of California, Davis

We discuss the construction and operation of a complex nonlinear optical system that uses ultrafast all-optical switching to isolate Raman from fluorescence signals. Using this system we are able to successfully separate Raman and fluorescence signals utilizing pulse energies and average powers that remain biologically safe.

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Biology

Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety
Bonnie Barrilleaux 1, Paul Knoepfler 1
1Department of Cell Biology and Human Anatomy, School of Medicine, University of California, Davis

Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.

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Immunology and Infection

Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field
John Y. Takekawa 1, Nichola J. Hill 1,2, Annie K. Schultz 1, Samuel A. Iverson 1, Carol J. Cardona 3,4, Walter M. Boyce 2, Joseph P. Dudley 5
1USGS Western Ecological Research Center, 2Wildlife Health Center, University of California, Davis, 3Department of Population Health and Reproduction, University of California, Davis, 4Department of Veterinary and Biomedical Sciences, University of Minnesota , 5Science Applications International Corporation

This study describes diagnosis of avian influenza in wild birds using a portable rRT-PCR system. The method takes advantage of freeze-dried reagents to screen wild birds in a non-laboratory setting, typical of an outbreak scenario. Use of molecular tools provides accurate and sensitive alternatives for rapid diagnosis.

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Neuroscience

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo
Benjamin Sadrian 1, Huaiyang Chen 1, Qizhi Gong 1
1Department of Cell Biology and Human Anatomy, School of Medicine, University of California, Davis

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

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Bioengineering

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments
Xiaoting Meng *1, Wenfei Li *2,3, Fraser Young 1, Runchi Gao 3, Laura Chalmers 3, Min Zhao 3, Bing Song 1
1School of Dentistry, Cardiff Institute of Tissue Engineering & Repair, Cardiff University , 2Shandong Qianfoshan Hospital, Shandong University School of Medicine, 3Dermatology and Ophthalmology Research, Institute for Regenerative Cures, University of California at Davis

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

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Biology

Quantifying Mixing using Magnetic Resonance Imaging
Emilio J. Tozzi 1, Kathryn L. McCarthy 1, Lori A. Bacca 2, William H. Hartt 2, Michael J. McCarthy 1
1Dept. Food Science and Technology, University of California, Davis, 2Corporate Engineering and Technology Laboratory, Procter & Gamble Company

Magnetic resonance imaging (MRI) provides a powerful tool to evaluate the effectiveness of process equipment during operation. We discuss the use of MRI to visualize mixing in a static mixer. The application is relevant to personal care products, but can be applied to a broad range of food, chemical, biomass and biological fluids.

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Neuroscience

Inducing Dendritic Growth in Cultured Sympathetic Neurons
Atefeh Ghogha 1, Donald A. Bruun 1, Pamela J. Lein 1
1Department of Molecular Biosciences, University of California, Davis

We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.

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Bioengineering

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix
Wenting Shih 1, Soichiro Yamada 1
1University of California, Davis

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

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Biology

Fruit Volatile Analysis Using an Electronic Nose
Simona Vallone 1, Nathan W. Lloyd 2, Susan E. Ebeler 3, Florence Zakharov 1
1Department of Plant Sciences, University of California, Davis, 2Department of Chemical Engineering and Material Science, University of California, Davis, 3Department of Viticulture and Enology, University of California, Davis

A rapid method for volatile compound analysis in fruit is described. The volatile compounds present in the headspace of a homogenate of the sample are rapidly separated and detected with ultra-fast gas chromatography (GC) coupled with a surface acoustic wave (SAW) sensor. A procedure for data handling and analysis is also discussed.

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JoVE Journal

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy
Chun A. Changou 1,2, Deanna L. Wolfson 2, Balpreet Singh Ahluwalia 2,3, Richard J. Bold 4,5, Hsing-Jien Kung 5,6, Frank Y.S. Chuang 1,2,5
1Department of Biochemistry and Molecular Medicine, University of California, Davis , 2NSF Center for Biophotonics Science & Technology, University of California, Davis , 3University of Tromsø, 4Department of Surgery (Division of Surgical Oncology), University of California, Davis , 5UC Davis Comprehensive Cancer Center, University of California, Davis , 6Department of Biological Chemistry, University of California, Davis

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

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JoVE Journal

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature
Andrew J. McElrone 1,2, Brendan Choat 3, Dilworth Y. Parkinson 4, Alastair A. MacDowell 4, Craig R. Brodersen 5
1U.S. Department of Agriculture, 2Department of Viticulture and Enology, University of California - Davis, 3Hawkesbury Institute for the Environment, University of Western Sydney, 4Advanced Light Source, Lawrence Berkeley National Lab, 5Citrus Research & Education Center, University of Florida

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

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JoVE Journal

Surface Renewal: An Advanced Micrometeorological Method for Measuring and Processing Field-Scale Energy Flux Density Data
Andrew J. McElrone 1,2, Thomas M. Shapland 2, Arturo Calderon 2,3, Li Fitzmaurice 4,5, Kyaw Tha Paw U 4, Richard L. Snyder 4
1Crops Pathology and Genetics Research Unit, United States Department of Agriculture-Agricultural Research Service, 2Department of Viticulture & Enology, University of California, Davis, 3Faculty of Agricultural Sciences, University of Chile, 4Atmospheric Science, University of California, Davis, 5URS Corporation Australia Pty. Ltd.

Surface renewal is a micrometeorological method that is being used increasingly to determine energy fluxes, but its technical complexity makes it inaccessible to a broad audience. We describe the steps needed to set up and calibrate a surface renewal field station, to acquire and process data, and to correctly interpret results.

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Medicine

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice: A Model for Immunotherapy Development
Daniel P. Vang 1, Gregory T. Wurz 1, Stephen M. Griffey 2, Chiao-Jung Kao 1, Audrey M. Gutierrez 1, Gregory K. Hanson 1, Michael Wolf 3, Michael W. DeGregorio 1
1Department of Internal Medicine, Division of Hematology and Oncology, University of California, Davis, 2Comparative Pathology Laboratory, UC Davis School of Veterinary Medicine, University of California, Davis, 3Merck Serono Research, Merck KGaA, Darmstadt, Germany

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

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Neuroscience

Electrophysiological Recording in the Brain of Intact Adult Zebrafish
Lindsey Johnston 1, Rebecca E. Ball 2, Seth Acuff 3, John Gaudet 4, Andrew Sornborger 5, James D. Lauderdale 2
1Department of Genetics, University of Georgia, 2Department of Cellular Biology, University of Georgia, 3College of Engineering, Architecture and Technology, Oklahoma State University, 4University of Georgia, 5Department of Mathematics, University of California, Davis

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

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Engineering

Real-Time DC-dynamic Biasing Method for Switching Time Improvement in Severely Underdamped Fringing-field Electrostatic MEMS Actuators
Joshua Small 1, Adam Fruehling 2, Anurag Garg 3, Xiaoguang Liu 1, Dimitrios Peroulis 3
1Department of Electrical and Computer Engineering, University of California, Davis, 2Digital Light Projection (DLP) Technology Development, Texas Instruments, 3Birck Nanotechnology Center and the Department of Electrical and Computer Engineering, Purdue University

The robust device design of fringing-field electrostatic MEMS actuators results in inherently low squeeze-film damping conditions and long settling times when performing switching operations using conventional step biasing. Real-time switching time improvement with DC-dynamic waveforms reduces the settling time of fringing-field MEMS actuators when transitioning between up-to-down and down-to-up states.

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Environment

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water
Bledar Bisha 1, Jaclyn A. Adkins 2, Jana C. Jokerst 3, Jeffrey C. Chandler 1, Alma Pérez-Méndez 4, Shannon M. Coleman 4, Adrian O. Sbodio 5, Trevor V. Suslow 5, Michelle D. Danyluk 6, Charles S. Henry 2, Lawrence D. Goodridge 7
1Department of Animal Science, University of Wyoming, 2Department of Chemistry, Colorado State University, 3Department of Environmental and Radiological Health Sciences, Colorado State University, 4Department of Animal Sciences, Colorado State University, 5Department of Plant Sciences, University of California, Davis, 6Department of Food Science and Human Nutrition, Citrus Research and Education Center, University of Florida, 7Department of Food Science and Agricultural Chemistry, McGill University

A protocol involving integrated concentration, enrichment, and end-point colorimetric detection of foodborne pathogens in large volumes of agricultural water is presented here. Water is filtered through Modified Moore Swabs (MMS), enriched with selective or non-selective media, and detection is performed using paper-based analytical devices (µPAD) imbedded with bacterial-indicative colorimetric substrates.

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Medicine

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene
Angel Alvarez 1, Gustavo A. Barisone 1, Elva Diaz 1
1Department of Pharmacology, University of California, Davis

The Focus Formation Assay provides a straightforward method to assess the transforming potential of a candidate oncogene.

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Immunology and Infection

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages
Jing Wu 1, Roberta Pugh 1, Richard C. Laughlin 1, Helene Andrews-Polymenis 2, Michael McClelland 3, Andreas J. Bäumler 4, L. Garry Adams 1
1Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, 2Department of Microbial and Molecular Pathogenesis, College of Medicine, Texas A&M University System Health Science Center, 3University of California, Irvine, 4Department of Medical Microbiology & Immunology, School of Medicine, University of California, Davis

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

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Medicine

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders
Dennis J. Wu 1, Neha Dixit 1, Erika Suzuki 1, Thanh Nguyen 2, Hyun Seock Shin 1, Jack Davis 2, Emanual Maverakis 3, Iannis E. Adamopoulos 1,2
1Division of Rheumatology, Allergy and Clinical Immunology, University of California, Davis, 2Institute for Pediatric Regenerative Medicine, Shriners Hospitals for Children - Northern California, 3Department of Dermatology, University of California, Davis

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

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Biology

Utilizing Custom-designed Galvanotaxis Chambers to Study Directional Migration of Prostate Cells
Hsin-ya Yang 1, Thi Dinh La 1, R. Rivkah Isseroff 1
1Department of Dermatology, Scool of Medicine, University of California, Davis

We present a method to apply a physiological electric field to migrating, immortalized prostate cells in a custom-made galvanotaxis chamber. Using this method, we demonstrate that 2 lines of non-tumorigenic prostate cells demonstrate different degrees of migration directionality in the field.

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Medicine

Calibrated Forceps Model of Spinal Cord Compression Injury
Ashley McDonough *1,2, Angela Monterrubio *1,2, Jeanelle Ariza 1,2, Verónica Martínez-Cerdeño 1,2
1Department of Pathology and Laboratory Medicine, University of California, Davis, 2Institute for Pediatric Regenerative Medicine, Shriners Hospitals for Children (Northern California)

Spinal cord injury models should be highly reproducible. We demonstrate that the calibrated forceps compression model of spinal cord injury is an easy to use surgical method for generating reproducible injuries to the murine spinal cord.

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JoVE Core

A Testing Platform for Durability Studies of Polymers and Fiber-reinforced Polymer Composites under Concurrent Hygrothermo-mechanical Stimuli
Antonio Gomez 1, Robert Pires 1, Alyssa Yambao 1, Valeria La Saponara 1
1Department of Mechanical and Aerospace Engineering, University of California, Davis

The durability of polymers and fiber-reinforced polymer composites in service is a critical aspect for their designs and condition-based maintenance. We present a novel low-cost laboratory testing platform for the investigation of the influence of concurrent mechanical and environmental loadings, and may help design more efficient yet safer composite structures.

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Medicine

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia
Yu Ouyang 1, Martin S. Judenhofer 1, Jeffrey H. Walton 1,2, Jan Marik 3, Simon P. Williams 3, Simon R. Cherry 1,4
1Department of Biomedical Engineering, University of California, Davis, 2Nuclear Magnetic Resonance Facility, University of California, Davis, 3Biomedical Imaging, Genentech, Inc, 4Department of Radiology, University of California, Davis

The method presented here uses simultaneous positron emission tomography and magnetic resonance imaging. In the cerebral hypoxia-ischemia model, dynamic changes in diffusion and glucose metabolism occur during and after injury. The evolving and irreproducible damage in this model necessitates simultaneous acquisition if meaningful multi-modal imaging data are to be acquired.

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Biology

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique
Guillaume Luxardi 1, Brian Reid 1, Fernando Ferreira 1,2, Pauline Maillard 3, Min Zhao 1,4
1Department of Dermatology, Institute for Regenerative Cures, University of California, Davis, 2Departamento de Biologia, Centro de Biologia Molecular e Ambiental, Universidade do Minho, 3Department of Neurology and Center for Neuroscience, University of California, Davis Imaging of Dementia and Aging Laboratory, 4Department of Ophthalmology, Institute for Regenerative Cures, University of California, Davis

Transporters in cell membranes allow differential segregation of ions across cell membranes or cell layers and play crucial roles during tissue physiology, repair and pathology. We describe the ion-selective self-referencing microelectrode that allows the measurement of specific ion fluxes at single cells and tissues in vivo.

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Education

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1
Demet Candas 1, Lili Qin 1, Ming Fan 1, Jian-Jian Li 1
1Radiation Oncology, University of California, Davis

Here, we outline how to study mitochondrial localization of a (cell cycle) kinase, and how to determine its sub-mitochondrial location as well as potential mitochondrial substrates/targets. Forced expression of proteins into the mitochondria provides a useful tool for studying the functional consequences of mitochondrial localization of a protein of interest.

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Immunology and Infection

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa
Xiangyang Shi 1, Hong Lin 2
1Department of Plant Science, University of California, Davis, 2Agricultural Research Service, San Joaquin Valley Agricultural Sciences Center, United States Department of Agriculture

In this study, a nano-microfluidic flow chamber was employed to visualize and functionally characterize the twitching motility of Xylella fastidiosa, a bacterium that causes Pierce's disease in grapevine.

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Genetics

Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes
Yanina S. Bogliotti 1, Marcela Vilarino 1, Pablo J. Ross 1
1Department of Animal Science, University of California, Davis

This protocol shows how to perform cytoplasmic microinjection in farm animal zygotes. This technique can be used to deliver any solution into the one-cell embryo such as genome editing tools to generate knockout animals.

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Genetics

The Use of Induced Somatic Sector Analysis (ISSA) for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development
Antanas Spokevicius 1, Lynette Taylor 1, Emma Melder 1, Kim Van Beveren 1, Josquin Tibbits 2, Nicky Creux 3,4, Gerd Bossinger 1
1School of Ecosystem and Forest Sciences, Faculty of Science, The University of Melbourne, 2Victorian AgriBiosciences Centre, La Trobe University R&D Park, 3College of Biological Sciences, Department of Plant Biology, University of California, Davis, 4Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria

Here we present a protocol that facilitates the medium to high throughput functional characterization of gene and promoter constructs in tree secondary stem tissue within comparatively short time frames. It is efficient, easy to use and widely applicable to a range of tree species.

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Biochemistry

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles
Yuanpei Li 1,2, Gaurav Bharadwaj 1, Joyce S. Lee 3
1Department of Biochemistry & Molecular Medicine, University of California, Davis, 2UC Davis Comprehensive Cancer Center, University of California, Davis, 3Department of Internal Medicine, Division of Hematology and Oncology, University of California, Davis

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

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Engineering

Spark Plasma Sintering Apparatus Used for the Formation of Strontium Titanate Bicrystals
Lauren A. Hughes 1, Klaus van Benthem 1
1Department of Materials Science and Engineering, University of California, Davis

A viable technique for the formation of strontium titanate bicrystals at high pressure and fast heating rate via the spark plasma sintering apparatus is developed.

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JoVE Journal

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model
Ann Lee-Barthel 1, Keith Baar 1,2, Daniel W. D. West 1,3
1Department of Neurobiology, Physiology, and Behavior, University of California, Davis, 2Department of Physiology and Membrane Biology, University of California, Davis, 3Faculty of Kinesiology and Physical Education, University of Toronto

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

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Environment

Vegetated Treatment Systems for Removing Contaminants Associated with Surface Water Toxicity in Agriculture and Urban Runoff
Brian S. Anderson 1,2, Bryn M. Phillips 1,2, Jennifer P. Voorhees 1,2, Michael Cahn 3
1Department of Environmental Toxicology, University of California, Davis, 2Marine Pollution Studies Laboratory - Granite Canyon, 3University of California, Cooperative Extension, Monterey County

This article summarizes the design attributes and the effectiveness of treatment systems that treat urban stormwater and agriculture irrigation runoff to remove pesticides and other contaminants associated with aquatic toxicity.

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Biology

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity
Wan-Shan Yang 1, Mel Campbell 2, Hsing-Jien Kung 2,3,4,5, Pei-Ching Chang 1,6
1Institute of Microbiology and Immunology, National Yang-Ming University, 2UC Davis Cancer Center, University of California, Davis, 3Department of Biochemistry and Molecular Medicine, University of California, Davis, 4Institute for Translational Medicine, College of Medical Science and Technology, Taipei Medical University, 5Division of Molecular and Genomic Medicine, National Health Research Institutes, 6Center for Infectious Disease and Cancer Research, Kaohsiung Medical University

Unlike ubiquitin ligases, few E3 SUMO ligases have been identified. This modified in vitro SUMOylation protocol is able to identify novel SUMO E3 ligases by an in vitro reconstitution assay.

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Biology

Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy
Christopher P. Chen 1, Frank Chuang 2, Yoshihiro Izumiya 1
1Department of Dermatology, University of California Davis School of Medicine, University of California, Davis, 2Center for Biophotonics, Department of Biochemistry and Molecular Medicine, University of California, Davis

Viral transcriptional factories are discrete structures that are enriched with cellular RNA polymerase II to increase viral gene transcription during reactivation. Here, a method to locate sites of actively transcribing viral chromatin in 3D nuclear space by a combination of immunofluorescence staining and in situ RNA hybridization is described.

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Immunology and Infection

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy
Ronald H.L. Li 1, Fern Tablin 2
1Department of Veterinary Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, 2Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California, Davis

We describe methods to isolate canine neutrophils from whole blood and visualize NET formation in live neutrophils using fluorescence microscopy. Also described are protocols to quantify NET formation and citrullinated histone H3 (citH3) expression using immunofluorescence microscopy.

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Immunology and Infection

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice
Yongli Zhang *1,2, Hangjie Zhang *2, Wenqiang Ma *3, Kefang Liu 1,2, Min Zhao 4, Yingze Zhao 2, Xuancheng Lu 5, Fuping Zhang 6, Xiangdong Li 3, George F. Gao 1,2,4,6, William J. Liu 1,2
1School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, 2NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, 3State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, 4Research Network of Immunity and Health (RNIH), Beijing Institutes of Life Science, Chinese Academy of Sciences, 5Laboratory Animal Center, Chinese Center for Disease Control and Prevention, 6CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

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Medicine

An In Vitro Approach to Photodynamic Therapy
Evan Austin 1,2, Jared Jagdeo 1,2,3
1Department of Dermatology, University of California, Davis, 2Dermatology Service, Sacramento VA Medical Center, 3Department of Dermatology, State University of New York, Downstate Medical Center

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce apoptosis. We present an in vitro PDT protocol designed to simulate PDT that may be used to study differences in PS incubation and light treatment parameters.

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Genetics

Purification and Transplantation of Myogenic Progenitor Cell Derived Exosomes to Improve Cardiac Function in Duchenne Muscular Dystrophic Mice
Xuan Su *1,2, Yan Shen *1, Yue Jin *1,2, Meng Jiang 2, Neal Weintraub 1, Yaoliang Tang 1
1Vascular Biology Center, Medical College of Georgia, Augusta University, 2Renji Hospital, School of Medicine, Shanghai Jiaotong University

Here, we present a protocol to transiently improve cardiac function in Duchenne muscular dystrophy mice by transplanting exosomes derived from normal myogenic progenitor cells.

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Developmental Biology

CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors
Yue Jin 1, Yan Shen 1, Xuan Su 1, Neal Weintraub 1, Yaoliang Tang 1
1Medical College of Georgia, Augusta University

Here, we present a Cas9-based exon23 deletion protocol to restore dystrophin expression in iPSC from Dmdmdx mouse-derived skin fibroblasts and directly differentiate iPSCs into myogenic progenitor cells (MPC) using the Tet-on MyoD activation system.

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Environment

PARbars: Cheap, Easy to Build Ceptometers for Continuous Measurement of Light Interception in Plant Canopies
William T. Salter 1, Andrew M. Merchant 1, Matthew E. Gilbert 2, Thomas N. Buckley 2
1School of Life and Environmental Sciences, Sydney Institute of Agriculture, University of Sydney, 2Department of Plant Sciences, University of California, Davis

Here, we present detailed instructions on how to build and calibrate research quality ceptometers (light sensors that integrate light intensity across many sensors arrayed linearly along a horizontal bar).

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Bioengineering

Prospecting Microbial Strains for Bioremediation and Probiotics Development for Metaorganism Research and Preservation
Helena D. M. Villela 1, Caren L. S. Vilela 1, Juliana M. Assis 1, Natascha Varona 2, Camille Burke 2, David A. Coil 2, Jonathan A. Eisen 2, Raquel S. Peixoto 1,2,3
1LEMM, Laboratory of Molecular Microbial Ecology, Institute of Microbiology Paulo de Góes, Federal University of Rio de Janeiro (UFRJ), 2Genome Center, University of California, Davis, 3IMAM-AquaRio - Rio de Janeiro Aquarium Research Center

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Bioremediation is the capacity of organisms to degrade contaminants. Here, we describe methodologies to isolate and test microbes presenting bioremediation ability and potential probiotic characteristics for corals.

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Genetics

Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes
Yana P. Blokhina 1,2, Ivan Olaya 1,2, Sean M. Burgess 1
1Department of Molecular and Cellular Biology, University of California, Davis, 2Integrative Genetics and Genomics Graduate Group, University of California, Davis

Nuclear surface spreads are an indispensable tool for studying chromosome events during meiosis. Here we demonstrate a method to prepare and visualize meiotic chromosomes during prophase I from zebrafish spermatocytes.

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Immunology and Infection

TurboID-Based Proximity Labeling for In Planta Identification of Protein-Protein Interaction Networks
Yongliang Zhang *1, Yuanyuan Li *2,3, Xinxin Yang 1, Zhiyan Wen 1, Ugrappa Nagalakshmi 2, Savithramma P. Dinesh-Kumar 2,3
1State Key Laboratory of Agro-Biotechnology and Ministry of Agriculture Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, 2Department of Plant Biology, College of Biological Sciences, University of California, Davis, 3Genome Center, College of Biological Sciences, University of California, Davis

Described here is a proximity labeling method for identification of interaction partners of the TIR domain of the NLR immune receptor in Nicotiana benthamiana leaf tissue. Also provided is a detailed protocol for the identification of interactions between other proteins of interest using this technique in Nicotiana and other plant species.

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Immunology and Infection

Identification of Neutrophil Extracellular Traps in Paraffin-Embedded Feline Arterial Thrombi using Immunofluorescence Microscopy
Laetitia Duler 1, Nghi Nguyen 2, Eric Ontiveros 3, Ronald H. L. Li 2
1Veterinary Medical Teaching Hospital, School of Veterinary Medicine, University of California, Davis, 2Department of Veterinary Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, 3Department of Veterinary Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis

We describe a method to identify neutrophil extracellular traps (NETs) in formaldehyde-fixed and paraffin-embedded feline cardiogenic arterial thrombi using heat-induced antigen retrieval and a double immunolabeling protocol.

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Biochemistry

Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins
David J. Speca 1, Elva Diaz 1
1Department of Pharmacology, UC Davis School of Medicine

Palmitoylation entails the incorporation of a 16-carbon palmitate moiety to cysteine residues of target proteins in a reversible manner. Here, we describe a biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest in mouse brain lysates.

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Determining the Influence of Soil Microbial Biomass Size on Soil Organic Matter Priming and Plant Residue Decomposition
Lu-Jun Li 1,2, Xia Zhu-Barker 3, Rongzhong Ye 4, William R. Horwath 3
1National Field Observation and Research Station of Hailun Agroecosystems, Key Laboratory of Mollisols Agroecology, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, 2University of Chinese Academy of Sciences, 3Department of Land, Air and Water Resources, University of California, Davis, 4Department of Plant & Environmental Sciences, Pee Dee Research & Education Center, Clemson University

This protocol describes a method to determine the influence of ryegrass residue addition on soil organic matter mineralization (i.e., priming effect) as well as explore the changes in soil microbial biomass size induced by soil organic matter priming, which involves artificially changing the size of microbial biomass.

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Biochemistry

Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers
Mowei Zhou 1, Shadan H. Abdali 1, David Dilworth 2, Lifeng Liu 2, Benjamin Cole 2, Neha Malhan 1, Amir H. Ahkami 1, Tanya E. Winkler 1, Joy Hollingsworth 3, Julie Sievert 3, Jeff Dahlberg 3, Robert Hutmacher 4,5, Mary Madera 6, Judith A. Owiti 6, Kim K. Hixson 1, Peggy G. Lemaux 6, Christer Jansson 1, Ljiljana Paša-Tolić 1
1Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 2DOE-Joint Genome Institute, Lawrence Berkeley Laboratory, 3Kearney Agricultural Research and Extension Center, University of California Agriculture and Natural Resources, 4West Side Research and Extension Center, University of California, 5Department of Plant Sciences, University of California, Davis, 6Department of Plant and Microbial Biology, University of California, Berkeley

The protocol has been developed to effectively extract intact histones from sorghum leaf materials for profiling of histone post-translational modifications that can serve as potential epigenetic markers to aid engineering drought resistant crops.

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Chemistry

Gold Nanoparticle Synthesis
Jonathan Marrs 1, Taher Ghomian 2, Lucas Domulevicz 1, Cliff McCold 3, Joshua Hihath 1
1Department of Electrical and Computer Engineering, University of California, Davis, 2Department of Computer Sciences and Electrical Engineering, Marshall University, 3Department of Materials Science and Engineering, University of California, Davis

A protocol for synthesizing ~12 nm diameter gold nanoparticles (Au nanoparticles) in an organic solvent is presented. The gold nanoparticles are capped with oleylamine ligands to prevent agglomeration. The gold nanoparticles are soluble in organic solvents such as toluene.

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Biology

A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing
Tse-Yu Chen 1, Adam E. Vorsino 2, Kyle J. Kosinski 1, Ana L. Romero-Weaver 1, Eva A. Buckner 1, Joanna C. Chiu 3, Yoosook Lee 1
1Florida Medical Entomology Laboratory, Department of Entomology and Nematology, Institute of Food and Agricultural Sciences, University of Florida, 2U. S. Fish and Wildlife Service, Pacific Islands Fish and Wildlife Office, 3Department of Entomology and Nematology, University of California Davis

Described here is a DNA extraction protocol using magnetic beads to produce high quality DNA extractions from mosquitoes. These extractions are suitable for a downstream next-generation sequencing approach.

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Neuroscience

AAV Deployment of Enhancer-Based Expression Constructs In Vivo in Mouse Brain
Tracy L. Warren 1,2, Jason T. Lambert 1,2, Alex S. Nord 1,2
1Department of Psychiatry and Behavioral Sciences, University of California, Davis, 2Department of Neurobiology, Physiology and Behavior, University of California, Davis

This protocol describes a novel rAAV-based transient enhancer-reporter assay. This assay can be used to induce enhancer-driven expression in vivo in the mouse brain.

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Immunology and Infection

Bacteriophage Effectiveness for Biocontrol of Foodborne Pathogens Evaluated via High-Throughput Settings
Yan D. Niu 1, Jae Eun Hyun 1, Nghi Nguyen 1
1Department of Ecosystem and Public Health, Faculty of Veterinary Medicine, University of Calgary

This protocol describes a robust method for using high-throughput settings to screen the antibacterial efficacy of bacteriophage cocktails.

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Bioengineering

Microfluidic Fabrication of Core-Shell Microcapsules carrying Human Pluripotent Stem Cell Spheroids
Kihak Gwon *1, Hye Jin Hong *1, Alan M. Gonzalez-Suarez 1, Gulnaz Stybayeva 1, Alexander Revzin 1
1Department of Physiology and Biomedical Engineering, Mayo Clinic

This article describes encapsulation of human pluripotent stem cells (hPSCs) using a co-axial flow focusing device. We demonstrate that this microfluidic encapsulation technology enables efficient formation of hPSC spheroids.

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Biochemistry

Quantifying the Binding Interactions Between Cu(II) and Peptide Residues in the Presence and Absence of Chromophores
Sohee Choi 1, Jessica A. San Juan 2, Marie C. Heffern 2, Michael J. Stevenson 1
1Department of Chemistry, University of San Francisco, 2Department of Chemistry, University of California, Davis

This article focuses on the use of electronic absorption spectroscopy and isothermal titration calorimetry to probe and quantify the thermodynamics of Cu(II) binding to peptides and proteins.

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Biology

Inducing Polyp Bail-out in Coral Colonies to Obtain Individualized Micropropagates for Laboratory Experimental Use
Pedro M. Cardoso 1, Ahmed A. Alsaggaf 1, Helena M. Villela 1, Raquel S. Peixoto 1
1Red Sea Research Center (RSRC), Division of Biological and Environmental Science and Engineering (BESE), King Abdullah University of Science and Technology (KAUST)

Polyp bail-out is a process induced by acute stress, in which coral polyps digest the tissue connecting them to their colony and detach from it to live as individuals. The present protocol describes how to induce coral micropropagation by bail-out using hypersaline or calcium-free seawater treatments.

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Genetics

Development of Knock-Out Muscle Cell Lines using Lentivirus-Mediated CRISPR/Cas9 Gene Editing
Mathilde Beaufils 1, Amandine Tourel 1, Anne Petiot 1, Nicole B. Halmai 2, Dave J. Segal 2, John Rendu 1, Isabelle Marty 1
1University Grenoble Alpes, INSERM, U1216, CHU Grenoble Alpes, Grenoble Institut Neurosciences, 2Genome Center, University of California, Davis

The protocol describes how to generate knock-out myoblasts using CRISPR/Cas9, starting from the design of guide-RNAs to the cellular cloning and characterization of the knock-out clones.

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Genetics

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae
Diedre Reitz 1, Jérôme Savocco 2, Aurèle Piazza 2, Wolf-Dietrich Heyer 1,3
1Department of Microbiology & Molecular Genetics, University of California, Davis, 2Laboratory of Biology & Modeling of the Cell, École Normale Supérieure de Lyon, 3Department of Molecular & Cellular Biology, University of California, Davis

The D-loop capture (DLC) and D-loop extension (DLE) assays utilize the principle of proximity ligation together with quantitative PCR to quantify D-loop formation, D-loop extension, and product formation at the site of an inducible double-stranded break in Saccharomyces cerevisiae.

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Genetics

High-Efficiency Gene Disruption in Primary Bone Marrow-Derived Macrophages Using Electroporated Cas9-sgRNA Complexes
Julia Craft 1, Tina Truong 1, Bennett H. Penn 1,2
1Department of Internal Medicine, University of California, Davis, 2Department of Medical Microbiology and Immunology, University of California, Davis

This protocol describes the procedure for genome editing in mouse bone marrow-derived macrophages using Cas9-sgRNA ribonucleoprotein complexes assembled in vitro and delivered by electroporation.

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