University of California, Los Angeles View Institution's Website 122 articles published in JoVE Developmental Biology Using Ex Vivo Live Imaging to Investigate Cell Divisions and Movements During Mouse Dental Renewal Abinaya Sundari Thooyamani*1, Elias Shahin*2, Sanako Takano1, Amnon Sharir2, Jimmy K. Hu1,3 1School of Dentistry, University of California Los Angeles, 2The Institute of Biomedical and Oral Research, Faculty of Dental Medicine, Hebrew University of Jerusalem, 3Molecular Biology Institute, University of California Los Angeles Ex vivo live imaging is a powerful technique for studying the dynamic processes of cellular movements and interactions in living tissues. Here, we present a protocol that implements two-photon microscopy to live track dental epithelial cells in cultured whole adult mouse incisors. Biochemistry A Knowledge Graph Approach to Elucidate the Role of Organellar Pathways in Disease via Biomedical Reports Alexander R. Pelletier1,2,3, Dylan Steinecke1,3,4, Dibakar Sigdel1, Irsyad Adam1, J. Harry Caufield1, Vladimir Guevara-Gonzalez1, Joseph Ramirez1, Aarushi Verma1, Kaitlyn Bali1, Katherine Downs1, Wei Wang1,2,3, Alex Bui3,4, Peipei Ping1,2,3,4,5 1Department of Physiology, UCLA School of Medicine, 2Scalable Analytics Institute (ScAi) at Department of Computer Science, UCLA School of Engineering, 3NIH BRIDGE2AI Center at UCLA & NHLBI Integrated Cardiovascular Data Science Training Program, UCLA, 4Medical Informatics, University of California at Los Angeles (UCLA), 5Department of Medicine (Cardiology), UCLA School of Medicine A computational protocol, CaseOLAP LIFT, and a use case are presented for investigating mitochondrial proteins and their associations with cardiovascular disease as described in biomedical reports. This protocol can be easily adapted to study user-selected cellular components and diseases. Immunology and Infection Oral Combinational Antiretroviral Treatment in HIV-1 Infected Humanized Mice Wenli Mu1, Anjie Zhen1, Mayra A. Carrillo1, Valerie Rezek1, Heather Martin1, Miguel Lizarraga1, Scott Kitchen1 1Division of Hematology and Oncology, David Geffen School of Medicine, University of California, Los Angeles This protocol describes a novel method to deliver oral combinational antiretroviral drugs that successfully suppress HIV-1 RNA replication in humanized mice. Bioengineering Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models Jesse Liang1, Alireza Sohrabi1, Mary Epperson1, Laila M. Rad1, Kelly Tamura1, Mayilone Sathialingam1, Thamira Skandakumar1, Philip Lue1, Jeremy Huang1, James Popoli1, Aidan Yackly1, Michael Bick1, Ze Zhong Wang1, Chia-Chun Chen1, Grigor Varuzhanyan1, Robert Damoiseaux1, Stephanie K. Seidlits1 1University of California Los Angeles The present protocol describes an experimental platform to assess the effects of mechanical and biochemical cues on chemotherapeutic responses of patient-derived glioblastoma cells in 3D matrix-mimetic cultures using a custom-made UV illumination device facilitating high-throughput photocrosslinking of hydrogels with tunable mechanical features. Bioengineering Engineering Antiviral Agents via Surface Plasmon Resonance Irene Maier1,2 1Department of Environmental Health Sciences, University of California, Los Angeles, 2Department of General Surgery, Medical University of Vienna The present protocol describes new tools for SPR binding assays to examine CV-N binding to HA, S glycoprotein, related hybrid-type glycans, and high-mannose oligosaccharides. SPR is used to determine the KD for binding either dimeric or monomeric CV-N to these glycans. Biology Quantitating Iron Transport Across the Mouse Placenta In Vivo Using Nonradioactive Iron Isotopes Veena Sangkhae1, Elizabeta Nemeth1 1Center for Iron Disorders, David Geffen School of Medicine, University of California, Los Angeles This article demonstrates how to prepare and administer transferrin-bound nonradioactive isotopic iron for studies of iron transport in mouse pregnancy. The approach for quantifying isotopic iron in fetoplacental compartments is also described. Bioengineering Simplified, High-throughput Analysis of Single-cell Contractility using Micropatterned Elastomers Lara Hairapetian1, Enrico Cortes1, Junyi Zhao1, Yao Wang1, Ricky Huang1, Robert Damoiseaux1,2, Ivan Pushkarsky1 1Forcyte Biotechnologies, 2University of California, Los Angeles This work presents a flexible protocol for utilizing fluorescently labeled elastomeric contractible surfaces (FLECS) Technology in microwell format for simplified, hands-off quantification of single-cell contractile forces based on visualized displacements of fluorescent protein micropatterns. Biology The Use of the Patch-Clamp Technique to Study the Thermogenic Capacity of Mitochondria Ambre M. Bertholet1 1Department of Physiology, David Geffen School of Medicine, University of California Los Angeles This method article details the main steps in measuring H+ leak across the inner mitochondrial membrane with the patch-clamp technique, a new approach to study the thermogenic capacity of mitochondria. Medicine Focal Laser Ablation of Prostate Cancer: An Office Procedure Wayne G. Brisbane1, Shyam Natarajan1, Alan Priester1, Ely R. Felker1, Adam Kinnaird2, Leonard S. Marks1 1University of California Los Angeles, 2University of Alberta This article presents and describes an outpatient treatment for prostate cancer using focal laser ablation. Laser catheter placement is guided by MRI-ultrasound fusion imaging in a fashion similar to prostate needle biopsy. Treatment is monitored in real-time with a thermal probe, placed adjacent to the laser fiber. Biochemistry Microcrystal Electron Diffraction of Small Molecules Michael W. Martynowycz1,2, Tamir Gonen1,2,3 1Howard Hughes Medical Institute, University of California Los Angeles, 2Department of Biological Chemistry, University of California Los Angeles, 3Department of Physiology, University of California Los Angeles Here, we describe the procedures developed in our laboratory for preparing powders of small molecule crystals for microcrystal electron diffraction (MicroED) experiments. Chemistry Optimization of Radiochemical Reactions using Droplet Arrays Alejandra Rios1,2, Travis S. Holloway2,3, Jia Wang2,4, R. Michael van Dam1,2,3,4 1Physics and Biology in Medicine Interdepartmental Graduate Program, University of California Los Angeles (UCLA), 2Crump Institute of Molecular Imaging, UCLA, 3Department of Molecular & Medical Pharmacology, David Geffen School of Medicine, 4Department of Bioengineering, UCLA This method describes the use of a novel high-throughput methodology, based on droplet chemical reactions, for the rapid and economical optimization of radiopharmaceuticals using nanomole amounts of reagents. Cancer Research A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth David Jelinek1,2, Ellen Ran Zhang1,2,3, Aaron Ambrus1,2, Erin Haley3, Emily Guinn1,2, Austin Vo1,2, Peter Le1,2, Ayse Elif Kesaf1,2, Jennifer Nguyen1,2, Lily Guo1,2, Destiny Frederick1,2, Zhengyang Sun1,2, Natalie Guo3, Parker Sevier1,2, Eric Bilotta1,2, Kaiser Atai1,2,4, Laurent Voisin1,2, Hilary A. Coller1,2,4 1Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, 2Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, 3Department of Molecular Biology, Princeton University, 4Molecular Biology Institute, University of California, Los Angeles A protocol to co-inject cancer cells and fibroblasts and monitor tumor growth over time is provided. This protocol can be used to understand the molecular basis for the role of fibroblasts as regulators of tumor growth. Biology Injection of Hydrogel Biomaterial Scaffolds to The Brain After Stroke Katrina L. Wilson1, S. Thomas Carmichael2, Tatiana Segura1,3,4 1Department of Biomedical Engineering, Duke University, 2Department of Neurology, University of California Los Angeles, 3Department of Neurology, Duke University, 4Department of Dermatology, Duke University Stroke is a global issue with minimal treatment options and no current clinical therapy for regenerating the lost brain tissue. Here we describe methods for creating precise photothrombotic stroke in the motor cortex of rodents and subsequent injection of hydrogel biomaterials to study their effects on tissue regeneration after stroke. Medicine In Vivo Mouse Model of Spinal Implant Infection Benjamin V. Kelley1, Christopher Hamad1, Stephen D. Zoller1, Danielle Greig1, Zeinab Mamouei1, Rene Chun1, Kellyn Hori1, Nicolas Cevallos1, Chad Ishmael1, Peter Hsiue1, Rishi Trikha1, Troy Sekimura2, Brandon Gettleman3, Autreen Golzar2, Adrian Lin2, Thomas Olson2, Ameen Chaudry2, Michael M. Le2, Anthony A. Scaduto1, Kevin P. Francis1, Nicholas M. Bernthal1 1Department of Orthopaedic Surgery, University of California Los Angeles, 2David Geffen School of Medicine, University of California Los Angeles, 3University of South Carolina School of Medicine, University of South Carolina The protocol describes a novel in vivo mouse model of spinal implant infection where a stainless-steel k-wire implant is infected with bioluminescent Staphylococcus aureus Xen36. Bacterial burden is monitored longitudinally with bioluminescent imaging and confirmed with colony forming unit counts after euthanasia. Medicine Novel Percutaneous Approach for Deployment of 3D Printed Coronary Stenosis Implants in Swine Models of Ischemic Heart Disease John J. Hollowed1,2, Caroline M. Colbert3,4, Jesse W. Currier1,2, Kim-Lien Nguyen1,2,3,4 1Division of Cardiology, David Geffen School of Medicine, University of California Los Angeles, 2VA Greater Los Angeles Healthcare System, U.S. Department of Veterans Affairs, 3Physics and Biology in Medicine Graduate Program, University of California Los Angeles, 4Diagnostic Cardiovascular Imaging Laboratory, Department of Radiological Sciences, David Geffen School of Medicine, University of California Los Angeles We describe a novel, cost-effective, and efficient technique for percutaneous delivery of three-dimensionally printed coronary implants to create closed-chest swine models of ischemic heart disease. The implants were fixed in place using a mother-and-child extension catheter with high success rate. Cancer Research Comparing Metastatic Clear Cell Renal Cell Carcinoma Model Established in Mouse Kidney and on Chicken Chorioallantoic Membrane Moe Ishihara*1, Junhui Hu*1, Xiaoyu Zhang2, YongHyeon Choi3, Anthony Wong4, Celine Cano-Ruiz5, Rongwei Zhao6, Ping Tan7, Jonathan L. Tso1, Lily Wu1,8 1Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, 2Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, 3Department of Bioengineering, Hanyang University, 4Department of Ecology and Evolutionary Biology, University of California, Los Angeles, 5Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, 6School of Life Sciences, Beijing Normal University, 7Department of Urology, West China Hospital, Sichuan University, 8Department of Urology, David Geffen School of Medicine, University of California, Los Angeles Metastatic clear cell renal cell carcinoma is a disease without a comprehensive animal model for thorough preclinical investigation. This protocol illustrates two novel animal models for the disease: the orthotopically implanted mouse model and the chicken chorioallantoic membrane model, both of which demonstrate lung metastasis resembling clinical cases. Cancer Research Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts Sanghee Lee1,2, Danielle N. Burner1,2, Theresa R. Mendoza1,2, Michelle T. Muldong1,2, Catalina Arreola1,2, Christina N. Wu2,3, Nicholas A. Cacalano4, Anna A. Kulidjian2,5, Christopher J. Kane1,2, Christina A. M. Jamieson1,2 1Department of Urology, University of California, San Diego, 2Moores Cancer Center, University of California, San Diego, 3Department of Medicine, University of California, San Diego, 4Department of Radiation Oncology, University of California, Los Angeles, 5Department of Orthopedic Surgery, University of California, San Diego Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC. Cancer Research Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers Allison C. Sharrow1, Moe Ishihara1, Junhui Hu1, Il Hyun Kim1, Lily Wu1 1Molecular and Medical Pharmacology, University of California Los Angeles We present the chicken chorioallantoic membrane model as an alternative, transplantable, in vivo model for the engraftment of gynecological and urological cancer cell lines and patient-derived tumors. Cancer Research Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture Preston D. Crowell*1, Jenna M. Giafaglione*1, Takao Hashimoto2, Johnny A. Diaz2, Andrew S. Goldstein2,3,4,5,6 1Molecular Biology Interdepartmental Program, University of California, Los Angeles, 2Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, 3Department of Urology, David Geffen School of Medicine, University of California, Los Angeles, 4Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, 5Jonsson Comprehensive Cancer Center, University of California, Los Angeles, 6Molecular Biology Institute, University of California, Los Angeles Mouse prostate organoids represent a promising context to evaluate mechanisms that regulate differentiation. This paper describes an improved approach to establish prostate organoids, and introduces methods to (1) collect protein lysate from organoids, and (2) fix and stain organoids for whole-mount confocal microscopy. Biology Human Adipose Tissue Micro-fragmentation for Cell Phenotyping and Secretome Characterization Bianca Vezzani1,2, Mario Gomez-Salazar1, Joan Casamitjana1, Carlo Tremolada3, Bruno Péault1,4 1MRC Center for Regenerative Medicine, University of Edinburgh, 2Dept. of Morphology, Surgery and Experimental Medicine, Section of General Pathology, University of Ferrara, 3Italian Image Institute, 4Orthopaedic Hospital Research Center and Broad Stem Cell Research Center, David Geffen School of Medicine, University of California Here, we present human adipose tissue enzyme-free micro-fragmentation using a closed system device. This new method allows the obtainment of sub-millimeter clusters of adipose tissue suitable for in vivo transplantation, in vitro culture, and further cell isolation and characterization. Neuroscience Visualizing Astrocyte Morphology Using Lucifer Yellow Iontophoresis Stefanie L. Moye1, Blanca Diaz-Castro1, Mohitkumar R. Gangwani1, Baljit S. Khakh1,2 1Department of Physiology, David Geffen School of Medicine, University of California Los Angeles, 2Department of Neurobiology, David Geffen School of Medicine, University of California Los Angeles Astrocytes are morphologically complex cells, exemplified by their multiple processes and bushy territories. To analyze their elaborate morphology, we present a reliable protocol to perform intracellular Lucifer yellow iontophoresis in lightly fixed tissue. Medicine Use of MRI-ultrasound Fusion to Achieve Targeted Prostate Biopsy Rajiv Jayadevan1, Steve Zhou1, Alan M. Priester1, Merdie Delfin1, Leonard S. Marks1 1Department of Urology, University of California Los Angeles Presented herein is a protocol to perform targeted biopsy of the prostate using an MRI-ultrasound fusion system. Genetics Overexpressing Long Noncoding RNAs Using Gene-activating CRISPR Carl Robert Rankin1, Janet Treger1, Emmanuelle Faure-Kumar2, Jihane Benhammou1, Deborah Anisman-Posner3, Alex Edward Bollinger3, Charalabos Pothoulakis1, David Miguel Padua1 1Vatche and Tamar Manoukian Division of Digestive Diseases, Department of Medicine, University of California, Los Angeles, 2Vatche and Tamar Manoukian Division of Digestive Diseases, Integrated Molecular Technologies (IMT) Core, Department of Medicine, University of California, Los Angeles, 3Division of Hematology and Oncology, Department of Medicine, University of California, Los Angeles Traditional cDNA-based overexpression techniques have a limited applicability for the overexpression of long noncoding RNAs due to their multiple splice forms with potential functionality. This review reports a protocol using CRISPR technology to overexpress multiple splice variants of a long noncoding RNA. Medicine Cloud-Based Phrase Mining and Analysis of User-Defined Phrase-Category Association in Biomedical Publications Dibakar Sigdel*1,2, Vincent Kyi*1,2, Aiden Zhang*1, Shaun P. Setty3, David A. Liem1,2,4, Yu Shi5, Xuan Wang5, Jiaming Shen5, Wei Wang1,6,7, JiaWei Han5, Peipei Ping1,2,4,6 1The NIH BD2K Center of Excellence in Biomedical Computing, University of California, Los Angeles, 2Department of Physiology, University of California, Los Angeles, 3Department of Pediatric and Adult Congenital Heart Surgery, Miller Children's and Women's Hospital and Long Beach Memorial Hospital, 4Department of Medicine/Cardiology, University of California, Los Angeles, 5NIH BD2K Program Centers of Excellence for Big Data Computing -- KnowEng Center, Department of Computer Science, University of Illinois at Urbana-Champaign (UIUC), 6Scalable Analytics Institute (ScAi), University of California, Los Angeles, 7Department of Computer Science, University of California, Los Angeles We present a protocol and associated programming code as well as metadata samples to support a cloud-based automated identification of phrases-category association representing unique concepts in user selected knowledge domain in biomedical literature. The phrase-category association quantified by this protocol can facilitate in depth analysis in the selected knowledge domain. Cancer Research Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice Gilbert Gastelum1, Eric Y. Chang2, David Shackleford3, Nicholas Bernthal3, Jeffery Kraut1,3, Kevin Francis4, Victoria Smutko1, Patrick Frost1,3 1Greater Los Angeles Veteran Administration Healthcare System, 2San Diego Veterans Administration Healthcare System, 3University of California, Los Angeles, 4Perkin Elmer Here we use bioluminescent, X-ray, and positron-emission tomography/computed tomography imaging to study how inhibiting mTOR activity impacts bone marrow-engrafted myeloma tumors in a xenograft model. This allows for physiologically relevant, non-invasive, and multimodal analyses of the anti-myeloma effect of therapies targeting bone marrow-engrafted myeloma tumors in vivo. Bioengineering Meso-Scale Particle Image Velocimetry Studies of Neurovascular Flows In Vitro Ryan A. Peck*1, Edver Bahena*1, Reza Jahan2, Guillermo Aguilar1,3,4, Hideaki Tsutsui1,4, Marko Princevac1, Monica M. Wilhelmus1, Masaru P. Rao1,3,4 1Department of Mechanical Engineering, University of California, Riverside, 2Division of Interventional Neuroradiology, University of California, Los Angeles, 3Materials Science and Engineering Program, University of California, Riverside, 4Department of Bioengineering, University of California, Riverside Here we present simplified methods for fabricating transparent neurovascular phantoms and characterizing the flow therein. We highlight several important parameters and demonstrate their relationship to field accuracy. Chemistry Automation of a Positron-emission Tomography (PET) Radiotracer Synthesis Protocol for Clinical Production Eric Schopf*1, Christopher M. Waldmann*2,3, Jeffrey Collins2,4, Christopher Drake1, Roger Slavik2,3, R. Michael van Dam2,4 1SOFIE, 2Department of Molecular & Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles (UCLA), 3Ahmanson Translational Imaging Division, University of California, Los Angeles (UCLA), 4Crump Institute for Molecular Imaging, University of California, Los Angeles (UCLA) Positron-emission tomography (PET) imaging sites that are involved in multiple early clinical research trials need robust and versatile radiotracer manufacturing capabilities. Using the radiotracer [18F]Clofarabine as an example, we illustrate how to automate the synthesis of a radiotracer using a flexible, cassette-based radiosynthesizer and validate the synthesis for clinical use. Behavior Stress-Enhanced Fear Learning, a Robust Rodent Model of Post-Traumatic Stress Disorder Abha K. Rajbhandari1,2, Sarah T. Gonzalez1,2, Michael S. Fanselow1,2,3 1Department of Psychology, University of California, Los Angeles, 2Staglin Center for Brain and Behavioral Health, University of California, Los Angeles, 3Department of Psychiatry and Biobehavioral Sciences, University of California, Los Angeles Here we describe the detailed methodology required to conduct stress-enhanced fear learning (SEFL) experiments, a preclinical model of post-traumatic stress disorder, in both rats and mice. The model utilizes aspects of Pavlovian fear conditioning and freezing as an index of enhanced fear in rodents. Behavior Continuous Theta Burst Stimulation of the Posterior Medial Frontal Cortex to Experimentally Reduce Ideological Threat Responses Colin Holbrook1, Chelsea L. Gordon1, Marco Iacoboni2 1Department of Cognitive and Information Sciences, University of California, Merced, 2Department of Psychiatry and Biobehavioral Sciences, University of California, Los Angeles Threats reliably evoke shifts in high-level ideological investment, but little work to date has explored the neural mechanisms underlying these dynamics. This paper describes how continuous theta burst transcranial magnetic stimulation may be employed to test the contribution of the posterior medial frontal cortex (and/or other regions) to threat-related ideological shifts. Medicine A Metadata Extraction Approach for Clinical Case Reports to Enable Advanced Understanding of Biomedical Concepts John Harry Caufield1,2, David A. Liem1,2,3, Anders O. Garlid1,2, Yijiang Zhou4, Karol Watson1,3, Alex A. T. Bui1,5,6,7, Wei Wang1,7,8,9, Peipei Ping1,2,3,7,8 1The NIH BD2K Center of Excellence in Biomedical Computing, University of California, Los Angeles, 2Department of Physiology, University of California, Los Angeles, 3Department of Medicine/Cardiology, University of California, Los Angeles, 4Department of Cardiology, First Affiliated Hospital, Zhejiang University School of Medicine, 5Department of Radiological Sciences, University of California, Los Angeles, 6Department of Bioengineering, University of California, Los Angeles, 7Scalable Analytics Institute (ScAi), University of California, Los Angeles, 8Department of Bioinformatics, University of California, Los Angeles, 9Department of Computer Science, University of California, Los Angeles We present a protocol and associated metadata template for the extraction of text describing biomedical concepts in clinical case reports. The structured text values produced through this protocol can support deep analysis of thousands of clinical narratives. Bioengineering Light-sheet Fluorescence Microscopy for the Study of the Murine Heart Yichen Ding1, Zachary Bailey2, Victoria Messerschmidt2, Jun Nie3, Richard Bryant2, Sandra Rugonyi4, Peng Fei3, Juhyun Lee1,2, Tzung K. Hsiai1 1Department of Bioengineering, University of California Los Angeles, 2Department of Bioengineering, University of Texas at Arlington, 3School of Optical and Electronic Information, Huazhong University of Science and Technology, 4Department of Biomedical Engineering, OSHU This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart. Bioengineering Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells Weikun Xiao1, Arshia Ehsanipour1, Alireza Sohrabi1, Stephanie K. Seidlits2 1Department of Bioengineering, University of California, Los Angeles, 2Department of Bioengineering, Jonsson Comprehensive Cancer Center, Broad Stem Cell Research Center, Brain Research Institute, University of California, Los Angeles Here, we present a protocol for three-dimensional culture of patient-derived glioblastoma cells within orthogonally tunable biomaterials designed to mimic the brain matrix. This approach provides an in vitro, experimental platform that maintains many characteristics of in vivo glioblastoma cells typically lost in culture. Bioengineering Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart Victoria Messerschmidt*1, Zachary Bailey*1, Kyung In Baek2, Yichen Ding2, Jeffrey J. Hsu2, Richard Bryant1, Rongsong Li3, Tzung K. Hsiai2, Juhyun Lee1 1Department of Bioengineering, The University of Texas at Arlington, 2Department of Medicine (Cardiology) and Bioengineering, UCLA, 3College of Health Science and Environmental Engineering, Shenzhen Technology University Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation. Cancer Research Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer Milica Momcilovic1, Sean T. Bailey2, Jason T. Lee3, Charles Zamilpa3, Anthony Jones3, Gihad Abdelhady1, James Mansfield4, Kevin P. Francis5, David B. Shackelford1 1Division of Orthopaedic Surgery, University of California Los Angeles David Geffen School of Medicine In this protocol, we describe how to utilize [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography (18F-FDG PET/CT) imaging to measure the tumor metabolic response to the targeted therapy MLN0128 in a Kras/Lkb1 mutant mouse model of lung cancer and coupled imaging with high resolution ex vivo autoradiography and quantitative histology. Biochemistry Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue David Jelinek1,2, Aimee Flores1,3, Melanie Uebelhoer1, Vincent Pasque2, Kathrin Plath2,3, M. Luisa Iruela-Arispe1,3, Heather R. Christofk2,3, William E. Lowry1,3, Hilary A. Coller1,2,3 1Department of Molecular, Cell and Developmental Biology, UCLA, 2Department of Biological Chemistry, David Geffen School of Medicine, 3Molecular Biology Institute Interdepartmental Program, UCLA We describe a protocol for mapping the spatial distribution of enzymatic activity for enzymes that generate nicotinatmide adenine dinucleotide phosphate (NAD(P)H) + H+ directly in tissue samples. Behavior Assessing Activity-based Anorexia in Mice Amanda C. Welch1, William R. Katzka1,2, Stephanie C. Dulawa1 1Department of Psychiatry, University of California, San Diego, 2University of California, Los Angeles Mice individually housed with a running wheel while given limited access to food develop reductions in food consumption and increase activity on the running wheel. This experimental phenomenon is called activity-based anorexia. This paradigm provides an experimental tool for studying the neurobiology and behaviors underlying aspects of anorexia nervosa. Neuroscience Making, Testing, and Using Potassium Ion Selective Microelectrodes in Tissue Slices of Adult Brain J. Christopher Octeau1, Guido Faas2, Istvan Mody1,2, Baljit S. Khakh1,3 1Department of Physiology, David Geffen School of Medicine, University of California Los Angeles, 2Department of Neurology, David Geffen School of Medicine, University of California Los Angeles, 3Department of Neurobiology, David Geffen School of Medicine, University of California Los Angeles Potassium ions contribute to the resting membrane potential of cells and extracellular K+ concentration is a crucial regulator of cellular excitability. We describe how to make, calibrate and use monopolar K+-selective microelectrodes. Using such electrodes enables the measurement of electrically evoked K+ concentration dynamics in adult hippocampal slices. Developmental Biology Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System Marlin Touma1,2 1Neonatal/Congenital Heart Laboratory, Cardiovascular Research Laboratory, David Geffen School of Medicine, University of California, Los Angeles, 2Children's Discovery and Innovation Institute, Department of Pediatrics, David Geffen School of Medicine, University of California, Los Angeles High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo. Immunology and Infection A Method for Targeted 16S Sequencing of Human Milk Samples Nicole H. Tobin1, Cora Woodward1, Sara Zabih1, David J. Lee1, Fan Li1, Grace M. Aldrovandi1 1Department of Pediatrics, University of California at Los Angeles A semi-automated workflow is presented for targeted sequencing of 16S rRNA from human milk and other low-biomass sample types. Behavior A Simple and Low-cost Assay for Measuring Ambulation in Mouse Models of Muscular Dystrophy Elizabeth M. Gibbs1,2, Rachelle H. Crosbie-Watson1,2,3,4 1Department of Integrative Biology and Physiology, University of California, 2Center for Duchenne Muscular Dystrophy, University of California, 3Department of Neurology, David Geffen School of Medicine, University of California, 4Molecular Biology Institute, University of California This protocol describes a flexible, low-cost system for measuring mouse ambulation in an open field activity assay. We show that a 6-minute ambulation assay based on this system detects a decrease in voluntary movement in mdx mice, and accurately distinguishes improvement in a muscle-specific rescue of these animals. Immunology and Infection High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection Kristel Lourdault1,2, James Matsunaga1,2, Karen V. Evangelista1,2, David A. Haake1,2,3,4 1Veterans Affairs Greater Los Angeles Healthcare System, 2Departments of Medicine, David Geffen School of Medicine at University of California Los Angeles, 3Departments of Urology, David Geffen School of Medicine at University of California Los Angeles, 4Departments of Microbiology, Immunology, and Molecular Genetics, University of California Los Angeles We describe here a technique that combines transposon mutagenesis with high-throughput sequencing to identify and quantify transposon leptospiral mutants in tissues after a challenge of hamsters. This protocol can be used to screen mutants for survival and dissemination in animals and can also be applied to in vitro studies. Immunology and Infection Quantification of Monocyte Transmigration and Foam Cell Formation from Individuals with Chronic Inflammatory Conditions Thomas A. Angelovich1,2, Anna C. Hearps1,3, Anna Maisa1, Theodoros Kelesidis4, Anthony Jaworowski1,3 1Centre for Biomedical Research, Burnet Institute, 2School of Health and Biomedical Sciences, RMIT University, 3Department of Infectious Diseases, Monash University, 4University of California, Los Angeles We describe a protocol to measure transmigration by monocytes across human endothelial monolayers and their subsequent maturation into foam cells. This provides a versatile method to assess the atherogenic properties of monocytes isolated from people with different disease conditions and to evaluate factors in blood which may enhance this propensity. Immunology and Infection A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity Fadi Kandarian1, Gemalene M Sunga1, Diana Arango-Saenz1, Maura Rossetti1 1UCLA Immunogenetics Center, Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here. Genetics Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow Gajendra W. Suryawanshi*1,2, Song Xu*3, Yiming Xie1, Tom Chou3, Namshin Kim4, Irvin S. Y. Chen1,5, Sanggu Kim6 1UCLA AIDS Institute, University of California at Los Angeles (UCLA), 2Department of Microbiology, Immunology, & Molecular Genetics, University of California at Los Angeles (UCLA), 3Departments of Biomathematics and Mathematics, University of California at Los Angeles (UCLA), 4Personalized Genomic Medicine Research Center, Division of Strategic Research Groups, Korea Research Institute of Bioscience and Biotechnology, 5Department of Medicine, University of California at Los Angeles (UCLA), 6Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University (OSU) This manuscript describes the experimental procedure and software analysis for a bidirectional integration site assay that can simultaneously analyze upstream and downstream vector-host junction DNA. Bidirectional PCR products can be used for any downstream sequencing platform. The resulting data are useful for a high-throughput, quantitative comparison of integrated DNA targets. Biology Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks Michelle Bradley1, Ivan Ramirez1, Keith Cheung1, Ankur A. Gholkar1, Jorge Z. Torres1,2,3 1Department of Chemistry and Biochemistry, University of California, Los Angeles, 2Molecular Biology Institute, University of California, Los Angeles, 3Jonsson Comprehensive Cancer Center, University of California, Los Angeles We describe a method for generating localization and affinity purification (LAP)-tagged inducible stable cell lines for investigating protein function, spatiotemporal subcellular localization and protein-protein interaction networks. Neuroscience An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach Sachin Parikh1, Andrew Le1, Julian Davenport1, Michael B. Gorin1, Steven Nusinowitz1, Anna Matynia1 1Jules Stein Eye Institute, University of California, Los Angeles Subretinal injections are the most common technique for delivering large therapeutic agents such as proteins and viral vectors to photoreceptors and the retinal pigment epithelium. An alternative method in mice that successfully targets the subretinal space with minimal collateral damage and fast recovery times is described here. Medicine Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model Aditya S. Shirali1, Austin I. McDonald2, Julia J. Mack3, M. Luisa Iruela-Arispe3 1Department of Surgery, University of California, Los Angeles, 2Molecular Biology Institute, University of California, Los Angeles, 3Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models. Developmental Biology Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue James E. Baily1, William C.W. Chen2,3, Nusrat Khan4, Iain R. Murray4, Zaniah N. González Galofre4, Johnny Huard5,6, Bruno Péault4,7 1Centre for Cardiovascular Science, Queen's Medical Research Institute, University of Edinburgh, 2Department of Bioengineering and Orthopaedic Surgery, University of Pittsburgh, 3Research Laboratory of Electronics and Department of Biological Engineering, Massachusetts Institute of Technology, 4MRC Centre for Regenerative Medicine, University of Edinburgh, 5Stem Cell Research Center, Department of Orthopaedic Surgery, University of Pittsburgh, 6Department of Orthopaedic Surgery, University of Texas Health Science Center at Houston, 7Department of Orthopaedic Surgery, UCLA Orthopaedic Hospital, David Geffen School of Medicine, University of California at Los Angeles Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium. Biology Working with Auditory HEI-OC1 Cells Gilda M. Kalinec1, Channy Park1, Pru Thein1, Federico Kalinec1 1Head and Neck Surgery, David Geffen School of Medicine, University of California, Los Angeles House Ear Institute-Organ of Corti 1 (HEI-OC1) is one of the few mouse auditory cell lines currently available for research purposes. This protocol describes how to work with HEI-OC1 cells to investigate the cytotoxic effects of pharmacological drugs as well as functional properties of inner ear proteins. Immunology and Infection Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons Deisy Contreras1, Vaithilingaraja Arumugaswami1,2,3 1Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, 2Department of Surgery, Cedars-Sinai Medical Center, 3Department of Surgery, David Geffen School of Medicine, University of California, Los Angeles Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed. Bioengineering Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery Raymond Wing Moon Lam1, Sunny Akogwu Abbah2, Wang Ming1, Mathanapriya Naidu1, Felly Ng1, Hu Tao1, James Goh Cho Hong3,4, Kang Ting5, Wong Hee Kit1,4 1Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, 2Centre for Research in Medical Devices (CÚRAM), National University of Ireland Galway, 3Department of Bioengineering, Faculty of Engineering, National University of Singapore, 4Tissue Engineering Program, National University of Singapore, 5Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, University of California, Los Angeles Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs. Medicine Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model Anjie Zhen1, Valerie Rezek1, Cindy Youn1, Jonathan Rick1, Brianna Lam1, Nelson Chang1, Jerome Zack1, Masakazu Kamata1, Scott Kitchen1 1David Geffen School of Medicine, University of California, Los Angeles This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection. Neuroscience Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY Dustin G. Roberts1, Hadley B. Johnsonbaugh1, Rory D. Spence2, Allan MacKenzie-Graham1 1Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, 2W.M. Keck Science Department, Claremont McKenna, Pitzer & Scripps Colleges Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results. Developmental Biology Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells Andrei A. Kramerov1, Mehrnoosh Saghizadeh1,2, Alexander V. Ljubimov1,2 1Eye Program, Board of Governors Regenerative Medicine Institute, Departments of Biomedical Sciences and Neurosurgery, Cedars-Sinai Medical Center, 2David Geffen School of Medicine, University of California, Los Angeles An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures. Medicine A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology Stefanie Nunez1, M. Mehdi Doroudchi1, Amy J. Gleichman1, Kwan L. Ng1, Irene L. Llorente1, Elif G. Sozmen1, S. Thomas Carmichael1, Jason D. Hinman1 1Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles Here we present methodology for the production of a focal stroke in murine white matter by local injection of an irreversible endothelial nitric oxide synthase (eNOS) inhibitor (L-Nio). Presented are two stereotactic variations, retrograde neuronal tracing, and fresh tissue labeling and dissection that expand the potential applications of this technique. Behavior Measurement of Neurophysiological Signals of Ignoring and Attending Processes in Attention Control Agatha Lenartowicz1, Gregory V. Simpson2, Samantha R. O'Connell1, Mark S. Cohen3 1Department of Psychiatry, University of California Los Angeles, 2Attention Research Institute, 3Departments of Psychiatry, Radiology, Neurology, Biomedical Physics, Psychology and Bioengineering, University of California Los Angeles Attention control comprises enhancement of target signals and attenuation of distractor signals. We describe an approach to measure separately but concurrently, the neurophysiology of attending and ignoring in sustained intermodal attention, utilizing a passive control condition during which neither process is continuously engaged. Bioengineering Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts Natalie L. Leong1, Nima Kabir1, Armin Arshi1, Azadeh Nazemi2, Ben M. Wu2, David R. McAllister1, Frank A. Petrigliano1 1Department of Orthopaedic Surgery, University of California Los Angeles, 2Department of Bioengineering, University of California Los Angeles Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model. Developmental Biology Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans Daniela A. Parodi1, Robert Damoiseaux3, Patrick Allard1,2 1Institute for Society and Genetics, University of California, Los Angeles, 2Environmental Health Sciences, University of California, Los Angeles, 3California Nanosystems Institute, Department of Molecular and Medical Pharmacology, University of California, Los Angeles We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos. Bioengineering Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement (EFIRM) Michael Tu1, Fang Wei1, Jieping Yang2, David Wong1 1School of Dentistry, University of California, Los Angeles, 2School of Medicine, Clinical Nutrition, University of California, Los Angeles Exosomes are microvesicular structures found within biofluids that potentially carry important disease discriminatory biomarkers. Here, a novel method is used to specifically extract exosomes and rapidly test the exosomal cargo for both RNA/protein targets following the disruption of exosomes using non-uniform electric cyclic square waves. Biology Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions Jason P. Awe1, Agustin Vega-Crespo1, James A. Byrne1,2 1Department of Molecular and Medical Pharmacology, University of California, Los Angeles (UCLA), 2Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles (UCLA) We describe a protocol for deriving lentiviral-based reprogrammed and characterized factor-free human induced pluripotent stem cells and conversion into putative clinical-grade conditions. Neuroscience Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators Ruotian Jiang1, Martin D. Haustein1, Michael V. Sofroniew2, Baljit S. Khakh1,2 1Department of Physiology, University of California Los Angeles, 2Department of Neurobiology, University of California Los Angeles The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ. Biology Dissection and Mounting of Drosophila Pupal Eye Discs Joy S. Tea1, Albert Cespedes1, Daniel Dawson2, Utpal Banerjee1,3, Gerald B. Call2 1Department of Molecular, Cell and Developmental Biology (MCDB), University of California at Los Angeles (UCLA), 2Arizona College of Osteopathic Medicine (AZCOM), Midwestern University, 3MCDB, Broad Stem Cell Research Center, UCLA The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age. Neuroscience Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina Allison Sargoy1, Steven Barnes1,2,3, Nicholas C. Brecha1,2,4, Luis Pérez De Sevilla Müller1 1Department of Neurobiology, University of California, Los Angeles, 2Veterans Administration Greater Los Angeles Healthcare System, 3Departments of Physiology & Biophysics and Ophthalmology & Visual Sciences, Dalhousie University, 4Departments of Neurobiology and Medicine, Jules Stein Eye Institute, CURE-Digestive Diseases Research Center, David Geffen School of Medicine, University of California, Los Angeles Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons. Bioengineering A Microfluidic Technique to Probe Cell Deformability David J. Hoelzle1,2, Bino A. Varghese1,3, Clara K. Chan1, Amy C. Rowat1 1Department of Integrative Biology and Physiology, University of California, Los Angeles, 2Department of Aerospace and Mechanical Engineering, University of Notre Dame, 3Molecular Imaging Center, University of Southern California We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions. Biology Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle William C.W. Chen1, Arman Saparov2,3, Mirko Corselli4, Mihaela Crisan5, Bo Zheng6, Bruno Péault7,8, Johnny Huard9 1Stem Cell Research Center, Department of Bioengineering and Orthopedic Surgery, University of Pittsburgh, 2Department of Orthopedic Surgery, University of Pittsburgh, 3Nazarbayev University Research and Innovation System, Nazarbayev University, 4Department of Orthopaedic Surgery, UCLA Orthopaedic Hospital and the Orthopaedic Hospital Research Center, University of California at Los Angeles, 5Department of Cell Biology, Erasmus MC Stem Cell Institute, 6OHSU Center for Regenerative Medicine, Oregon Health & Science University, 7 Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia. Medicine Network Analysis of the Default Mode Network Using Functional Connectivity MRI in Temporal Lobe Epilepsy Zulfi Haneef1,2, Agatha Lenartowicz3, Hsiang J. Yeh4, Jerome Engel Jr.4, John M. Stern4 1Department of Neurology, Baylor College of Medicine, 2Neurology Care Line, Michael E. DeBakey VA Medical Center, 3Semel Institute for Neuroscience and Human Behavior, University of California, Los Angeles, 4Department of Neurology, University of California, Los Angeles The Default Mode Network (DMN) in Temporal Lobe Epilepsy (TLE) is analyzed in the resting state of the brain using seed-based functional connectivity MRI (fcMRI). Behavior Method for Simultaneous fMRI/EEG Data Collection during a Focused Attention Suggestion for Differential Thermal Sensation Pamela K. Douglas1,2, Maureen Pisani2, Rory Reid1, Austin Head2, Edward Lau2, Ebrahim Mirakhor3, Jennifer Bramen2, Billi Gordon2, Ariana Anderson2, Wesley T. Kerr2, Chajoon Cheong4, Mark S. Cohen1,2 1Neuropsychiatric Institute, University of California, Los Angeles, 2Laboratory of Neuroimaging Technology, University of California, Los Angeles, 3Yale School of Medicine, 4Korean Basic Science Institute We present a protocol for concurrent collection of EEG/fMRI data, and synchronized MR clock signal recording. We demonstrate this method using a unique paradigm whereby subjects receive ‘cold glove’ instructions during scanning, and EEG/fMRI data are recorded along with hand temperature measurements both before and after hypnotic induction. Biology Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue Hassan Khalil1, Wenxian Nie1, Robert A Edwards2, James Yoo1 1Department of Surgery, UCLA, 2Department of Pathology, UC Irvine The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation. Medicine Parabiosis in Mice: A Detailed Protocol Paniz Kamran1,2, Konstantina-Ioanna Sereti1,2, Peng Zhao1,2, Shah R. Ali3, Irving L. Weissman3, Reza Ardehali1,2 1Department of Medicine-Division of Cardiology, University of California, Los Angeles, 2Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, 3Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine Parabiotic joining of two organisms leads to the development of a shared circulatory system. In this protocol, we describe the surgical steps to form a parabiotic connection between a wild-type mouse and a constitutive GFP-expressing mouse. Biology Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates Min Zhu1, Sepideh Heydarkhan-Hagvall2, Marc Hedrick1, Prosper Benhaim3,4, Patricia Zuk3,5 1Cytori Therapeutics Inc, 2Division of Cardiac Surgery, Department of Surgery, David Geffen School of Medicine at UCLA, 3Division of Plastic and Reconstructive Surgery, Department of Surgery, David Geffen School of Medicine at UCLA, 4Department of Orthopedic Surgery, David Geffen School of Medicine at UCLA, 5Regenerative Bioengineering and Repair Laboratory, David Geffen School of Medicine at UCLA In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase. Medicine Bioluminescent Orthotopic Model of Pancreatic Cancer Progression Ming G. Chai1, Corina Kim-Fuchs1,2, Eliane Angst2, Erica K. Sloan1,3 1Monash Institute of Pharmaceutical Sciences, Monash University, 2Department of Visceral Surgery and Medicine, University of Bern, 3Cousins Center for Neuroimmunology, University of California Los Angeles Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging. Bioengineering Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors Jae Youn Hwang1, Daniel L. Farkas1, Lali K. Medina-Kauwe2,3 1Department of Biomedical Engineering, University of Southern California, 2Department of Biomedical Sciences, Cedars-Sinai Medical Center, 3Geffen School of Medicine, University of California, Los Angeles This article details the procedures for optical imaging analysis of the tumor-targeted nanoparticle, HerDox. In particular, detailed use of the multimode imaging device for detecting tumor targeting and assessing tumor penetration is described here. Neuroscience Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish Yali Zhao1, Nancy L. Wayne1 1Department of Physiology, University of California, Los Angeles In this video, we will demonstrate how to record electrical activity from identified single neurons in a whole brain preparation, which preserves complex neural circuits. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with a fluorescent protein for identification in the intact brain preparation. Bioengineering Bacterial Detection & Identification Using Electrochemical Sensors Colin Halford1,2, Vincent Gau3, Bernard M. Churchill2, David A. Haake2,4,5 1Research Service, Veterans Affairs Greater Los Angeles Healthcare System, 2Department of Urology, The David Geffen School of Medicine, University of California, Los Angeles, 3GeneFluidics, 4Division of Infectious Diseases, Veterans Affairs Greater Los Angeles Healthcare System, 5Department of Microbiology, Immunology & Molecular Genetics, University of California, Los Angeles We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current. Medicine Remote Magnetic Navigation for Accurate, Real-time Catheter Positioning and Ablation in Cardiac Electrophysiology Procedures David Filgueiras-Rama1, Alejandro Estrada1, Josh Shachar2, Sergio Castrejón1, David Doiny1, Marta Ortega1, Eli Gang3, José L. Merino1 1Cardiology, Robotic Cardiac Electrophysiology and Arrhythmia Unit, La Paz University Hospital, 2Magnetecs Corp., 3Cardiology, Geffen School of Medicine at UCLA Los Angeles This report provides a detailed description of a new remote navigation system based on magnetic driven forces, which has been recently introduced as a new robotic tool for human cardiac electrophysiology procedures. Bioengineering Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone Hongying Zhu1, Aydogan Ozcan1,2,3 1Electrical Engineering Department, University of California, Los Angeles, 2Bioengineering Department, University of California, Los Angeles, 3California NanoSystems Institute (CNSI), University of California, Los Angeles We review our recent results on the integration of fluorescent microscopy and imaging flow cytometry tools on a cell-phone using compact and cost-effective opto-fluidic attachments. These cell-phone based micro-analysis devices might be useful for cytometric analysis, such as performing various cell counting tasks as well as for high-throughput screening of e.g., water samples in resource limited settings. Neuroscience Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures Bruce Yazejian1, Rita M. Yazejian1, Rachel Einarsson2, Alan D. Grinnell1 1Department of Physiology, David Geffen School of Medicine at UCLA, 2Natural Science Division, Pepperdine University This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings. Neuroscience Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato Ana Marie S. Palanca1, Alvaro Sagasti1 1Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles Optogenetic techniques have made it possible to study the contribution of specific neurons to behavior. We describe a method in larval zebrafish for activating single somatosensory neurons expressing a channelrhodopsin variant (ChEF) with a diode-pumped solid state (DPSS) laser and recording the elicited behaviors with a high-speed video camera. Biology Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein Riki Kawaguchi1, Ming Zhong1, Hui Sun1 1Department of Physiology, Jules Stein Eye Institute and Howard Hughes Medical Institute, University of California, Los Angeles Here we describe an optimized technique to produce high-quality vitamin A/RBP complex and two real-time monitoring techniques to study vitamin A transport by STRA6, the RBP receptor. Biology Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method (EFM) Lawren Sack1, Christine Scoffoni1 1University of California, Los Angeles We describe a relatively rapid (30 min) and realistic method for simultaneously measurement of leaf hydraulic conductance (Kleaf) and stomatal conductance (gs) for transpiring excised leaves. The method can be modified to measure the light and dehydration responses of Kleaf and gs. Medicine Quantitative Analysis of Chromatin Proteomes in Disease Emma Monte1, Haodong Chen1, Maria Kolmakova1, Michelle Parvatiyar1, Thomas M. Vondriska1,2,3, Sarah Franklin1,4 1Department of Anesthesiology, David Geffen School of Medicine at UCLA, 2Department of Medicine, David Geffen School of Medicine at UCLA, 3Department of Physiology, David Geffen School of Medicine at UCLA, 4Department of Internal Medicine, Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease. Immunology and Infection Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model Dimitrios N. Vatakis1,2,3, Gregory C. Bristol1,2, Sohn G. Kim1,2, Bernard Levin1,2, Wei Liu4, Caius G. Radu4, Scott G. Kitchen1,2,3, Jerome A. Zack1,2,5 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. Medicine Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate David D. Lo*1,2, Jeong S. Hyun*1,3, Michael T. Chung1, Daniel T. Montoro1, Andrew Zimmermann1, Monica M. Grova1,4, Min Lee5, Derrick C. Wan1, Michael T. Longaker1 1Department of Surgery, Stanford University, 2Department of Surgery, Duke University, 3Department of Surgery, Saint Joseph Mercy Hospital, 4School of Medicine, University of California, San Francisco, 5School of Dentistry, University of California, Los Angeles This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration. Immunology and Infection Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation Hon Wai Koon1, Samantha Ho1, Michelle Cheng1, Ryan Ichikawa1, Charalabos Pothoulakis1 1Center for Inflammatory Bowel Diseases, Division of Digestive Diseases, David Geffen School of Medicine, The University of California Los Angeles, Los Angeles Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype. Biology Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea Ahmed E. Hegab1, Vi Luan Ha1, Yasser S. Attiga1, Derek W. Nickerson1, Brigitte N. Gomperts1 1Department of Pediatrics, David Geffen School of Medicine at UCLA Here we demonstrate our protocol for isolation of basal and submucosal gland duct cells from mouse tracheas. We also demonstrate the method of injecting stem cells into the dorsal mouse fat pad to create an in vivo model of submucosal gland regeneration. Immunology and Infection Isolation of Lymphocytes from Mouse Genital Tract Mucosa Janina Jiang1, Kathleen A. Kelly1,2 1Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, 2California NanoSystems An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species Bioengineering Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution Serhan O. Isikman1, Waheb Bishara1, Aydogan Ozcan1,2,3 1Electrical Engineering Department, University of California, Los Angeles, 2Bioengineering Department, University of California, Los Angeles, 3California NanoSystems Institute, University of California, Los Angeles Lensfree optical tomography is a three-dimensional microscopy technique that offers a spatial resolution of <1 μm × <1 μm × <3 μm in x, y and z dimensions, respectively, over a large imaging-volume of 15-100 mm3, which can be particularly useful for integration with lab-on-a-chip platforms. Immunology and Infection Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus (MRSA) in Rat Wessam Abdel Hady1, Arnold S. Bayer1,2, Yan Q. Xiong1,2 1Department of Medicine, Division of Infectious Diseases, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, 2Geffen School of Medicine at UCLA Experimental rat endocarditis model due to methicillin-resistant S. aureus. Biology Aseptic Laboratory Techniques: Volume Transfers with Serological Pipettes and Micropipettors Erin R. Sanders1 1Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles When working in a laboratory, it is imperative to minimize sources of contamination. Aseptic technique refers to procedures that permit transfer of cultures and reagents while avoiding contact with non-sterile surfaces. Serological pipettes and micropipettors are used to measure precise volumes without compromising sterility of solutions used in experiments. Bioengineering Use of Human Perivascular Stem Cells for Bone Regeneration Aaron W. James*1, Janette N. Zara*2, Mirko Corselli2, Michael Chiang1, Wei Yuan2, Virginia Nguyen1, Asal Askarinam1, Raghav Goyal1, Ronald K. Siu3, Victoria Scott1, Min Lee3, Kang Ting1, Bruno Péault2,4, Chia Soo2 1Dental and Craniofacial Research Institute and Section of Orthodontics, School of Dentistry, UCLA, 2UCLA and Orthopaedic Hospital, Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, UCLA, 3Department of Bioengineering, UCLA, 4Center for Cardiovascular Science, University of Edinburgh Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo. Biology Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies Todd C. Lorenz1 1Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles PCR has emerged as a common technique in many molecular biology laboratories. Provided here is a quick guide to several conventional PCR protocols. Because each reaction is a unique experiment, optimal conditions required to generate a product vary. Understanding the variables in a reaction will greatly enhance troubleshooting efficiency, thereby increasing the chance to obtain the desired result. Biology Aseptic Laboratory Techniques: Plating Methods Erin R. Sanders1 1Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles When working with media and reagents used to culture microorganisms, aseptic technique must be practiced to ensure contamination is minimized. A variety of plating methods are routinely used to isolate, propagate, or enumerate bacteria and phage, all of which incorporate procedures that maintain the sterility of experimental materials. Biology Agarose Gel Electrophoresis for the Separation of DNA Fragments Pei Yun Lee1, John Costumbrado1, Chih-Yuan Hsu1, Yong Hoon Kim1 1Department of Molecular, Cell, and Developmental Biology, University of California Los Angeles A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described. Biology High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method Rachna Ujwal1, Jeff Abramson2 1UCLA-DOE Institute for Genomics and Proteomics, University of California Los Angeles, 2Department of Physiology, David Geffen School of Medicine, UCLA Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium. Medicine Mouse Eye Enucleation for Remote High-throughput Phenotyping Vinit B. Mahajan1,2, Jessica M. Skeie1,2, Amir H. Assefnia2,3, MaryAnn Mahajan1,2, Stephen H. Tsang2,4 1Department of Ophthalmology and Visual Sciences, University of Iowa, 2Omics Laboratory, University of Iowa, 3School of Dentistry, UCLA, 4Bernard and Shirlee Brown Glaucoma Laboratory, Department of Ophthalmology, College of Physicians and Surgeons, Columbia University The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens. Biology Isolation of CD133+ Liver Stem Cells for Clonal Expansion C. Bart Rountree1, Wei Ding1, Hein Dang1, Colleen VanKirk2, Gay M. Crooks3 1Department of Pediatrics and Pharmacology, Pennsylvania State College of Medicine, 2Department of Pharmacology, Pennsylvania State College of Medicine, 3Department of Pediatrics, University of California Los Angeles, School of Medicine Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion. Bioengineering Lensless Fluorescent Microscopy on a Chip Ahmet F. Coskun1, Ting-Wei Su1, Ikbal Sencan1, Aydogan Ozcan1 1Department of Electrical Engineering, University of California, Los Angeles A lensless on-chip fluorescent microscopy platform is demonstrated that can image fluorescent objects over an ultra-wide field-of-view of e.g., >0.6-8 cm2 with <4μm resolution using a compressive sampling based decoding algorithm. Such a compact and wide-field fluorescent on-chip imaging modality could be valuable for high-throughput cytometry, rare-cell research and microarray-analysis. Immunology and Infection Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins Marija Pinne1,2, David Haake3,4 1Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, 2Research service, 151, Veterans Affairs Greater Los Angeles Healthcare System, 3Departments of Medicine, Urology at David Geffen School of Medicine and Department of Microbiology, Immunology and Molecular Gentics, University of California Los Angeles (UCLA), 4Division of Infectious Diseases, 111F, Veterans Affairs Greater Los Angeles Health Care System An efficient method to assess surface-exposure of leptospiral proteins is described. The method is specifically designed to avoid disruption of the fragile outer membrane of leptospiral cells. This technique requires employment of several negative controls to assess the integrity of the outer membrane and specificity of antibody reaction. Biology Microwave-assisted One-pot Synthesis of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) Shuang Hou*1,2,3, Duy Linh Phung*1,2,3, Wei-Yu Lin1,2,3, Ming-wei Wang4, Kan Liu5, Clifton Kwang-Fu Shen1,2,3 1Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California at Los Angeles, 2Crump Institute for Molecular Imaging, David Geffen School of Medicine, University of California at Los Angeles, 3California NanoSystems Institute, University of California at Los Angeles, 4Nuclear Medicine, PET Center, Shanghai Medical Collegea, Fudan University, 5Electronics and Information Engineering, College of Electronics and Information Engineering, Wuhan Textile University A facile, one-pot synthesis of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) was developed based on a non-aqueous, three-step radiochemical process. Using microwave heating, the entire procedure can be completed in less than 30 min, or 60 min with further purification by preparative HPLC. The decay-corrected radiochemical yields (RCYs) were 35-5% (n > 30). Biology Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny Maureen R. Lynch1, Judith C. Gasson2, Helicia Paz1 1Department of Medicine, Hematology-Oncology, University of California, Los Angeles, 2Department of Biological Chemistry, University of California, Los Angeles mStrawberry OP9 cells allow for complete evaluation of all ES-derived progeny from co-culture. Neuroscience Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method Han-peng Xu1,2, Lin Gou3,4, Hong-Wei Dong3 1Department of Neurosurgery, Cedars Sinai Medical Center, UCLA, 2Basic Medicine School, Fourth Military Medical University, 3Department of Neurology, David Geffen School of Medicine, UCLA, 4Aerospace Medicine School, Fourth Military Medical Univeristy In this protocol, we described a new method to study the influence of glial cell heterogeneity on axon growth with an in vitro co-culture system. Rat cortical glial cells were cultured to confluence and cocultured with highly purified rat dorsal root ganglia neurons. Different glial cell influence on neurons adhesion and axon growth was compared directly in the same culture. This method provides a new way to directly study the glial cell heterogeneity influence on neuron adhesion and axon growth. Immunology and Infection In vivo Imaging of Transgenic Leishmania Parasites in a Live Host Colin J. Thalhofer1, Joel W. Graff2, Laurie Love-Homan3, Suzanne M. Hickerson4, Noah Craft5, Stephen M. Beverley4, Mary E. Wilson6,7 1Interdisciplinary Immunology Program, University of Iowa, and the VA Medical Center, 2Department of Biochemistry, University of Iowa, and the VA Medical Center, 3Department of Internal Medicine, University of Iowa, 4Department of Molecular Microbiology, Washington University School of Medicine, 5Division of Dermatology, Harbor-UCLA Medical Center, Hanley-Hardison Research Center, 6Interdisciplinary Immunology Program, Iowa City VA Medical Center, 7Departments of Internal Medicine, Microbiology and Epidemiology, University of Iowa An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection. Neuroscience Low-stress Route Learning Using the Lashley III Maze in Mice Amanda Bressler1, David Blizard2, Anne Andrews3,4,5,6 1Department of Chemistry, Pennsylvania State University, 2Center for Developmental and Health Genetics, Pennsylvania State University, 3Department of Veterinary and Biomedical Sciences, Pennsylvania State University, 4Huck Institute of the Life Sciences, Pennsylvania State University, 5California NanoSystems Institute, University of California, Los Angeles, 6Semel Institute of Neuroscience and Human Behavior, University of California, Los Angeles The Lashley III maze is a route-learning task that does not rely on aversive stimuli or visual cues. It is thus a highly attractive option for evaluating learning and memory, especially in aging mice or otherwise where stress is a consideration. Neuroscience Selection of Aptamers for Amyloid β-Protein, the Causative Agent of Alzheimer's Disease Farid Rahimi1, Gal Bitan1,2,3 1Department of Neurology, David Geffen School of Medicine, 2Molecular Biology Institute, University of California, Los Angeles, 3Brain Research Institute, University of California, Los Angeles Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid β-protein, the causative agent of Alzheimer's disease. Biology Lensless On-chip Imaging of Cells Provides a New Tool for High-throughput Cell-Biology and Medical Diagnostics Onur Mudanyali1, Anthony Erlinger1, Sungkyu Seo1, Ting-Wei Su1, Derek Tseng1, Aydogan Ozcan1,2 1Electrical Engineering Department, University of California, Los Angeles, 2California NanoSystems Institute, University of California, Los Angeles Lensfree on-chip imaging and characterization of cells is illustrated. This on-chip cell imaging approach provides a compact and cost-effective tool for medical diagnostics and high-throughput cell biology applications, making it especially suitable for resource poor settings. Biology DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation Marino DiFranco1, Marbella Quinonez1, Joana Capote1, Julio Vergara1 1Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy. Biology Preparation of Aplysia Sensory-motor Neuronal Cell Cultures Yali Zhao1, Dan O. Wang1, Kelsey C. Martin1,2,3 1Dept. of Psychiatry and Biobehavioral Sciences, University of California, Los Angeles, 2Dept. of Biological Chemistry, University of California, Los Angeles, 3Semel Institute for Neuroscience and Human Behavior, University of California, Los Angeles Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture. Biology Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels Harpreet Singh1, Sarah Warburton2, Thomas M. Vondriska3, Baljit S. Khakh1 1Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, 2Department of Anesthesiology, David Geffen School of Medicine, University of California, Los Angeles, 3Department of Anesthesiology, Medicine and Physiology, David Geffen School of Medicine, University of California, Los Angeles We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling. Biology Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes Eiji Shigetomi1, Baljit S. Khakh1 1Departments of Physiology and Neurobiology, David Geffen School of Medicine, University of California, Los Angeles We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy. Biology Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos Georgeann S. O'Brien1, Sandra Rieger1, Seanna M. Martin1, Ann M. Cavanaugh1, Carlos Portera-Cailliau2, Alvaro Sagasti1 1Department of Molecular Cell and Developmental Biology, University of California, Los Angeles, 2Departments of Neurology and Neurobiology, University of California, Los Angeles Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging. Biology Visually Mediated Odor Tracking During Flight in Drosophila Mark A. Frye1, Brian J. Duistermars1 1Department of Physiological Science, University of California, Los Angeles Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion. Biology Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides Farid Rahimi1, Panchanan Maiti1, Gal Bitan2,3 1Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, 2Brain Research Institute, Molecular Biology Institute, University of California, Los Angeles, 3Department of Neurology, University of California, Los Angeles Photo-induced cross-linking of unmodified proteins (PICUP) allows characterization of oligomer size distribution in metastable protein mixtures. We demonstrate application of PICUP to three representative amyloidogenic peptides the 40- and 42-residue forms of amyloid β-protein, and calcitonin, and a control peptide growth-hormone releasing factor. Biology A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila Brian J. Duistermars1, Mark A. Frye1 1Department of Physiological Science, University of California, Los Angeles Here we describe how to tether a fly in an olfactory magnetic-tether (OMT) apparatus. We describe how to align the rare-earth magnets and odor ports, and how to set mass flow rates for both the stimulus delivery and vacuum suction to achieve optimal odor tracking. Biology Isolation and Analysis of Hematopoietic Stem Cells from the Placenta Christos Gekas1, Katrin E. Rhodes1, Hanna K. A. Mikkola1 1Jonsson Comprehensive Cancer Center, University of California, Los Angeles We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta. Biology Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells Ivan Khvorostov1, Jin Zhang1, Michael Teitell1 1David Geffen School of Medicine, University of California, Los Angeles In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays. Biology From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel Jin Zhang1, Ivan Khvorostov1, Michael Teitell1 1David Geffen School of Medicine, University of California, Los Angeles This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 3 of 3. Biology From MEFs to Matrigel 2: Splitting hESCs from MEFs onto Matrigel Ivan Khvorostov1, Jin Zhang1, Michael Teitell1 1David Geffen School of Medicine, University of California, Los Angeles This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 2 of 3. Biology From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs Jin Zhang1, Ivan Khvorostov1, Michael Teitell1 1David Geffen School of Medicine, University of California, Los Angeles This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells. Part 1 of 3. Biology In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice Peyman Golshani1, Carlos Portera-Cailliau1 1Department of Neurology, University of California, Los Angeles To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . Biology A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window Ricardo Mostany1, Carlos Portera-Cailliau1 1Department of Neurology, University of California, Los Angeles Cortical blood flow dynamics can be studied in vivo by imaging fluorescent dextran dyes injected into the tail vein of rodents with 2-photon microscopy. This video shows how to image blood flow dynamics in neocortex of mice through a glass-covered cranial window preparation. Biology A Craniotomy Surgery Procedure for Chronic Brain Imaging Ricardo Mostany1, Carlos Portera-Cailliau1 1Department of Neurology, University of California, Los Angeles This video and protocol demonstrate how to implant a glass-covered cranial window in rodents. These preparations can be used for chronic in vivo two-photon imaging of the neocortex over time scales of months. It may also be used for other types of imaging, including optical intrinsic signal imaging. Biology Predicting the Effectiveness of Population Replacement Strategy Using Mathematical Modeling John Marshall1, Koji Morikawa1, Nicholas Manoukis1, Charles Taylor1 1Department of Ecology and Evolutionary Biology, University of California, Los Angeles Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy. Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies. The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.