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The Scripps Research Institute

39 ARTICLES PUBLISHED IN JoVE

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Biology

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates
Gregory M. Solis 1,2, Michael Petrascheck 1,2
1Department of Chemical Physiology, The Scripps Research Institute, 2Department of Molecular and Experimental Medicine, The Scripps Research Institute

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

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Biology

Crystallization of Membrane Proteins in Lipidic Mesophases
Wei Liu 1, Vadim Cherezov 1
1Molecular Biology, The Scripps Research Institute

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

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JoVE Journal

Flow Cytometry Purification of Mouse Meiotic Cells
Irina V. Getun 1, Bivian Torres 2, Philippe R.J. Bois 1
1Genome Plasticity Laboratory, Department of Cancer Biology, The Scripps Research Institute, 2Flow Cytometry Core, The Scripps Research Institute

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

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Biology

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts
Urszula Polak 1,2, Calley Hirsch 1, Sherman Ku 3, Joel Gottesfeld 3, Sharon Y.R. Dent 1, Marek Napierala 1
1Department of Molecular Carcinogenesis and Center for Cancer Epigenetics, University of Texas M.D. Anderson Cancer Center, 2Department of Cell Biology, Poznan University of Medical Sciences, 3Department of Molecular Biology, The Scripps Research Institute

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

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Immunology and Infection

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model
Dimitrios N. Vatakis 1,2,3, Gregory C. Bristol 1,2, Sohn G. Kim 1,2, Bernard Levin 1,2, Wei Liu 4, Caius G. Radu 4, Scott G. Kitchen 1,2,3, Jerome A. Zack 1,2,5
1Department of Medicine, Division of Hematology-Oncology, David Geffen School of Medicine at UCLA, 2UCLA AIDS Institute, 3Eli & Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, 4Department of Medical and Molecular Pharmacology, David Geffen School of Medicine at UCLA, 5Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine at UCLA

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

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Immunology and Infection

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells
Benson Y.H. Cheng *1, Emilio Ortiz-Riaño *1, Juan Carlos de la Torre 2, Luis Martínez-Sobrido 1
1Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, 2Departments of Immunology and Microbial Sciences, The Scripps Research Institute

Rescue of recombinant arenaviruses from cloned cDNAs, an approach referred to as reverse genetics, allows researchers to investigate the role of specific viral gene products, as well as the contribution of their different specific domains and residues, to many different aspects of the biology of arenavirus. Likewise, reverse genetics techniques in FDA-approved cell lines (Vero) for vaccine development provides novel possibilities for the generation of effective and safe vaccines to combat human pathogenic arenaviruses.

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Chemistry

A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators
Dejan Caglič 1, Kristin M. Bompiani 1, Michelle C. Krutein 1, Petr Čapek 1, Tobin J. Dickerson 1,2
1Department of Chemistry, The Scripps Research Institute, 2Worm Institute of Research and Medicine (WIRM), The Scripps Research Institute

The botulinum neurotoxin type A light chain (BoNT/A LC) is a metalloprotease that enters motor neurons, cleaves its substrate SNAP-25, and disrupts neurotransmission, thereby resulting in flaccid paralysis. Utilizing a high-throughput-compatible FRET-based assay, large libraries of small molecules can be screened for their impact on BoNT/A LC enzymatic activity.

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Bioengineering

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology
Xiaofeng Cui *1,2,3, Guifang Gao *2,4, Tomo Yonezawa 5,6, Guohao Dai 1
1Department of Biomedical Engineering, Rensselaer Polytechnic Institute, 2Stemorgan Inc., 3Institute of Advanced Study, Technical University of Munich, 4Institute of Virology, School of Medicine, Wuhan University, 5Department of Molecular and Experimental Medicine, The Scripps Research Institute, 6Research Institute for Biomedical Sciences, Tokyo University of Science

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

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JoVE Core

Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library
Yu Gao 1, Thomas Kodadek 1
1Scripps Florida, The Scripps Research Institute

Peptide tertiary amides (PTAs) are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. Here we describe a synthetic method which combines both split-and-pool and sub-monomer strategies to synthesize a one-bead one-compound library of PTAs.

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Biology

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro
Joseph Jablonski 1, Mark Clementz 1, Kevin Ryan 2, Susana T. Valente 1
1Department of Infectious Diseases, The Scripps Research Institute, 2Department of Chemistry, City College of New York

RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro.

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Biology

One-channel Cell-attached Patch-clamp Recording
Bruce A. Maki 1, Kirstie A. Cummings 2, Meaghan A. Paganelli 1, Swetha E. Murthy 3, Gabriela K. Popescu 4
1Graduate Program in Neuroscience, SMBS, University at Buffalo, SUNY, 2Department of Biochemistry, SMBS, University at Buffalo, SUNY, 3Molecular and Cellular Neuroscience Department, The Scripps Research Institute, 4Department of Biochemistry and Graduate Program in Neuroscience, SMBS, University at Buffalo, SUNY

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

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Neuroscience

Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis
Sylvia Neumann 1, George E. Campbell *1, Lukasz Szpankowski *2,3, Lawrence S.B. Goldstein 2,4, Sandra E. Encalada 1
1Department of Molecular and Experimental Medicine, Dorris Neuroscience Center, The Scripps Research Institute, 2Department of Cellular and Molecular Medicine, University of California San Diego, 3Department of Bioengineering, University of California San Diego, 4Department of Neurosciences, University of California San Diego School of Medicine

Intracellular transport of cargoes, such as vesicles or organelles, is carried out by molecular motor proteins that track on polarized microtubules. This protocol describes the correlation of the directionality of transport of individual cargo particles moving inside neurons, to the relative amount and type of associated motor proteins.

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Developmental Biology

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells
Peter Westenskow 1,2, Zack Sedillo 1,2, Ashley Barnett 2, Martin Friedlander 1,2
1Department of Cell and Molecular Biology, The Scripps Research Institute, 2Lowy Medical Research Institute

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

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Medicine

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension
Peter D. Westenskow 1,2, Toshihide Kurihara 1, Stephen Bravo 1, Daniel Feitelberg 1, Zack A. Sedillo 2, Edith Aguilar 1, Martin Friedlander 1,2
1Department of Cell and Molecular Biology, The Scripps Research Institute, 2Lowy Medical Research Institute

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

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Biology

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains
Thomas Gaj *1, Jia Liu *1,2
1Departments of Chemistry and Cell and Molecular Biology, The Scripps Research Institute, 2Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

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Neuroscience

Optogenetic Functional MRI
Peter Lin 1, Zhongnan Fang 2, Jia Liu 1, Jin Hyung Lee 1,2
1Neurology and Neurological Sciences, Stanford University, 2Electrical Engineering, Neurology and Neurological Sciences, Stanford University

This protocol describes the steps and data analysis required to successfully perform optogenetic functional magnetic resonance imaging (ofMRI). ofMRI is a novel technique that combines high-field fMRI readout with optogenetic stimulation, allowing for cell type-specific mapping of functional neural circuits and their dynamics across the whole living brain.

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Immunology and Infection

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins
Ryan McBride 1, James C. Paulson 1, Robert P. de Vries 1,2
1Department of Cell and Molecular Biology, Chemical Physiology and Microbial Science, The Scripps Research Institute, 2Department of Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

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Biology

Sequential Application of Glass Coverslips to Assess the Compressive Stiffness of the Mouse Lens: Strain and Morphometric Analyses
Catherine Cheng 1, David S. Gokhin 1, Roberta B. Nowak 1, Velia M. Fowler 1
1Department of Cell and Molecular Biology, The Scripps Research Institute

Age-related increases in eye lens stiffness are linked to presbyopia. This protocol describes a simple, cost-effective method for measuring mouse lens stiffness. Mouse lenses, like human lenses, become stiffer with age. This method is precise and can be adapted for lenses from larger animals.

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Biochemistry

Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography
Andrii Ishchenko 1,2, Vadim Cherezov 1,2, Wei Liu 3
1The Bridge Institute, University of Southern California, 2Department of Chemistry, University of Southern California, 3School of Molecular Sciences, Center for Applied Structural Discovery at the Biodesign Institute, Arizona State University

We describe procedures for the preparation and delivery of membrane protein microcrystals in lipidic cubic phase for serial crystallography at X-ray free-electron lasers and synchrotron sources. These protocols can also be applied for incorporation and delivery of soluble protein microcrystals, leading to substantially reduced sample consumption compared to liquid injection.

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Immunology and Infection

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay
Salome Murinello 1, Stacey K. Moreno 1, Matthew S. Macauley 2, Susumu Sakimoto 1, Peter D. Westenskow 1,3, Martin Friedlander 1
1Department of Cell and Molecular Biology, The Scripps Research Institute, 2Department of Chemical Physiology, The Scripps Research Institute, 3The Lowy Medical Research Institute

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

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Bioengineering

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening
Yaqian Li *1,2, Xiaojun Yan *1, Wei Liu *1, Lyu Zhou 1,3, Zhifeng You 1, Yanan Du 1,2
1Department of Biomedical Engineering, School of Medicine, Tsinghua University, 2Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, 3School of Life Sciences, Tsinghua University

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

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Biology

SA-β-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs
Heike Fuhrmann-Stroissnigg *1, Fernando E. Santiago *1,2,3, Diego Grassi 1, YuanYuan Ling 1, Laura J. Niedernhofer 1,2,3, Paul D. Robbins 1,2,3
1Department of Molecular Medicine and the Center on Aging, The Scripps Research Institute, 2Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 3Institute on the Biology of Aging and Metabolism, University of Minnesota

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated β-Galactosidase activity in single cells.

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Immunology and Infection

Antigenic Liposomes for Generation of Disease-specific Antibodies
Kyle J. Bednar *1, Lakeya Hardy *2,3, Johanna Smeekens *3,4, Dharmendra Raghuwanshi 5, Shiteng Duan 6, Mike D. Kulis 3,4, Matthew S. Macauley 5,7
1Janssen R&D, 2Department of Microbiology and Immunology, University of North Carolina, 3UNC Food Allergy Initiative, University of North Carolina, 4Department of Pediatrics, University of North Carolina, 5Department of Chemistry, University of Alberta, 6Department of Molecular Medicine, Scripps Research Institute, 7Department of Medical Microbiology and Immunology, University of Alberta

Described is the preparation of antigenic liposomal nanoparticles and their use in stimulating B-cell activation in vitro and in vivo. Consistent and robust antibody responses led to the development of a new peanut allergy model. The protocol for generating antigenic liposomes can be extended to different antigens and immunization models.

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Neuroscience

Inducement and Evaluation of a Murine Model of Experimental Myopia
Xiaoyan Jiang 1,2, Toshihide Kurihara 1,2, Shin-ichi Ikeda 1,2, Hiromitsu Kunimi 1,2, Kiwako Mori 1,2, Hidemasa Torii 1,2, Kazuo Tsubota 2
1Laboratory of Photobiology, Keio University School of Medicine, 2Department of Ophthalmology, Keio University School of Medicine

In this protocol, we describe the full process of experimental myopia inducement in mice using newly designed eyeglasses and the technic needed for achieving stable and reproducible results in ocular parameter measurements.

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Immunology and Infection

Merkel Cell Polyomavirus Infection and Detection
Wei Liu *1, Nathan A. Krump *1, Christopher B. Buck 2, Jianxin You 1
1Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, 2Laboratory of Cellular Oncology, National Cancer Institute

Here, we present a protocol to infect primary human dermal fibroblast with MCPyV. The protocol includes isolation of dermal fibroblasts, preparation of MCPyV virions, virus infection, immunofluorescence staining, and fluorescence in situ hybridization. This protocol can be extended for characterizing MCPyV-host interactions and discovering other cell types infectable by MCPyV.

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Genetics

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes
Jingxin Wang 1, John Hammond 2, Kristen A. Johnson 1
1Calibr, The Scripps Research Institute, 2Department of Integrative Structural and Computational Biology, The Scripps Research Institute

Detailed protocols for both in vitro and in-cell selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

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Biology

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands
Tatiana Zyrianova 1, Liana V. Basova 1, Helen Makarenkova 1
1Department of Molecular Medicine, The Scripps Research Institute

The lacrimal gland (LG) has two cell types expressing α-smooth muscle actin (αSMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

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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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Bioengineering

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy
Ying Yin *1, Siyu Wang *1, Danni Hu 1, Jingyao Cai 1, Fubin Chen 1, Bo Wang 1, Yu Gao 1
1Key Laboratory for Organic Electronics and Information Displays & Jiangsu Key Laboratory for Biosensors, Institute of Advanced Materials (IAM), Jiangsu National Synergistic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts & Telecommunications

Presented here is a protocol for the fabrication of iron oxide nanoparticle-shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform for magnetic hyperthermia and photothermal combination cancer therapy.

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JoVE Journal

RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points
Shubham Rathore 1, Jenni Hassert 1, Courtney M. Clark-Hachtel 2,3, Aaron Stahl 1,4, Yoshinori Tomoyasu 2, Elke K. Bushbeck 1
1Department of Biological Sciences, University of Cincinnati, 2Department of Biology, Miami University, 3Department of Biology, University of North Carolina at Chapel Hill, 4Department of Neuroscience, The Scripps Research Institute

RNA interference is a widely applicable, powerful technique for manipulating gene expression at specific developmental stages. Here, we describe the necessary steps for implementing this technique in the aquatic diving beetle Thermonectus marmoratus, from the acquisition of gene sequences to the knockdown of genes that affect structure or behavior.

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Chemistry

Quantitative SERS Detection of Uric Acid via Formation of Precise Plasmonic Nanojunctions within Aggregates of Gold Nanoparticles and Cucurbit[n]uril
Weng-I Katherine Chio 1, Gemma Davison 1, Tabitha Jones 1, Jia Liu 1, Ivan P. Parkin 1, Tung-Chun Lee 1,2
1Department of Chemistry, University College London (UCL), 2Institute for Materials Discovery, University College London (UCL)

A host-guest complex of cucurbit[7]uril and uric acid was formed in an aqueous solution before adding a small amount into Au NP solution for quantitative surface-enhanced Raman spectroscopy (SERS) sensing using a modular spectrometer.

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Medicine

A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion
Deokho Lee *1,2, Yukihiro Miwa *1,2,3, Heonuk Jeong 1,2, Shin-ichi Ikeda 1,2, Yusaku Katada 1,2, Kazuo Tsubota 1,2,4, Toshihide Kurihara 1,2
1Laboratory of Photobiology, Keio University School of Medicine, 2Department of Ophthalmology, Keio University School of Medicine, 3Animal eye care, Tokyo Animal Eye Clinic, 4Tsubota Laboratory, Inc.

Here, we describe a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion using simple sutures and a clamp. This model can be useful for understanding the pathological mechanisms of retinal ischemia caused by cardiovascular abnormalities.

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Bioengineering

Toxicity Screens in Human Retinal Organoids for Pharmaceutical Discovery
Kevin Eade 1,2, Sarah Giles 1,2, Sarah Harkins-Perry 1,2, Martin Friedlander 1,2
1Lowy Medical Research Institute, 2The Scripps Research Institute

Here we present a step-by-step protocol to generate mature human retinal organoids and utilize them in a photoreceptor toxicity assay to identify pharmaceutical candidates for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel).

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Biochemistry

Purification of Endogenous Drosophila Transient Receptor Potential Channels
Jia Liu *1, Yuyang Liu *1, Weidi Chen 1, Yuzhen Ding 1, Xiaoru Lan 1, Wei Liu 1
1Shenzhen Key Laboratory for Neuronal Structural Biology, Biomedical Research Institute, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center

Based on the assembling mechanism of the INAD protein complex, in this protocol, a modified affinity purification plus competition strategy was developed to purify the endogenous Drosophila TRP channel.

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Environment

A Low-Cost Method of Measuring the In Situ Primary Productivity of Periphyton Communities of Lentic Waters
Kateřina Čapková 1,3, Tomáš Bešta 1, Jan Mareš 1,2, Petr Čapek 2, Klára Řeháková 1,3
1Biology Centre of the CAS, Institute of Hydrobiology, 2Faculty of Science, University of South Bohemia, 3Institute of Botany AS CR

Presented here is a cost-effective and transportable method/facility for measuring the primary productivity of microbial mats under actual in situ environmental temperature and light conditions. The experimental setup is based on widely available materials and can be used under various conditions while offering the advantages of laboratory-based models.

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Biochemistry

Single-Particle Cryo-EM Data Collection with Stage Tilt using Leginon
Sriram Aiyer 1, Timothy S. Strutzenberg 1, Marianne E. Bowman 2, Joseph P. Noel 2,3, Dmitry Lyumkis 1,4,5
1Laboratory of Genetics, The Salk Institute for Biological Studies, 2Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies, 3Department of Chemistry and Biochemistry, University of California San Diego, 4Graduate School of Biological Sciences, Section of Molecular Biology, University of California San Diego, 5Department of Integrative Structural and Computational Biology, The Scripps Research Institute

The present protocol describes a generalized and easy-to-implement scheme for tilted single-particle data collection in cryo-EM experiments. Such a procedure is especially useful for obtaining a high-quality EM map for samples suffering from preferential orientation bias due to adherence to the air-water interface.

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Biology

Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis
Jinxi Yuan 1, Jie Zhang 1, Yan Zhang 1, WuYun QiQiGe 1, Wei Liu 2, Shanchun Yan 1, Guirong Wang 2
1Key Laboratory of Sustainable Forest Ecosystem Management - Ministry of Education, Northeast Forestry University, 2Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences

This paper presents the step-by-step protocols for CRISPR/Cas9 mutagenesis of the Oriental fruit fly Bactrocera dorsalis. Detailed steps provided by this standardized protocol will serve as a useful guide for generating mutant flies for functional gene studies in B. dorsalis.

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Neuroscience

Chronic Intermittent Ethanol Vapor Exposure Paired with Two-Bottle Choice to Model Alcohol Use Disorder
Tiange Xiao 1, Yueyi Chen 1, Alyssa Boisvert 1, Maury Cole 2, Adam Kimbrough 1,3,4,5
1Department of Basic Medical Sciences, College of Veterinary Medicine, Purdue University, 2The Scripps Research Institute, 3Purdue Institute for Integrative Neuroscience, 4Weldon School of Biomedical Engineering, Purdue University, 5Purdue Institute of Inflammation, Immunology, and Infectious Disease

Presented here is a protocol for the 2BC/CIE model of alcohol dependence in mice to study alcohol use disorder.

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Behavior

Quantifying Food Intake in Caenorhabditis elegans by Measuring Bacterial Clearance
Christina Clark *1,2,3, Alan To *1,2,3, Michael Petrascheck 1,2,3
1Department of Molecular and Cellular Biology, The Scripps Research Institute, 2Department of Molecular Medicine, The Scripps Research Institute, 3Department of Neuroscience, The Scripps Research Institute

This protocol describes an assay to quantify Caenorhabditis elegans feeding rate based on measuring the clearance of bacteria in liquid culture.

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