University of California, Los Angeles

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. Hu^{1,3 1}School 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. Pelletier^1,2,3, Dylan Steinecke^1,3,4, Dibakar Sigdel¹, Irsyad Adam¹, J. Harry Caufield¹, Vladimir Guevara-Gonzalez¹, Joseph Ramirez¹, Aarushi Verma¹, Kaitlyn Bali¹, Katherine Downs¹, Wei Wang^1,2,3, Alex Bui^3,4, Peipei Ping^{1,2,3,4,5 1}Department 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 Kitchen^{1 1}Division 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. Seidlits^{1 1}University 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 Maier^{1,2 1}Department 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 Nemeth^{1 1}Center 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 Damoiseaux^1,2, Ivan Pushkarsky^{1 1}Forcyte 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. Bertholet^{1 1}Department 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. Marks^{1 1}University 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. Martynowycz^1,2, Tamir Gonen^{1,2,3 1}Howard 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 Rios^1,2, Travis S. Holloway^2,3, Jia Wang^2,4, R. Michael van Dam^{1,2,3,4 1}Physics 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 Jelinek^1,2, Ellen Ran Zhang^1,2,3, Aaron Ambrus^1,2, Erin Haley³, Emily Guinn^1,2, Austin Vo^1,2, Peter Le^1,2, Ayse Elif Kesaf^1,2, Jennifer Nguyen^1,2, Lily Guo^1,2, Destiny Frederick^1,2, Zhengyang Sun^1,2, Natalie Guo³, Parker Sevier^1,2, Eric Bilotta^1,2, Kaiser Atai^1,2,4, Laurent Voisin^1,2, Hilary A. Coller^{1,2,4 1}Department 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 Segura^{1,3,4 1}Department 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. Bernthal^{1 1}Department 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. Hollowed^1,2, Caroline M. Colbert^3,4, Jesse W. Currier^1,2, Kim-Lien Nguyen^{1,2,3,4 1}Division 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 Wu^{1,8 1}Department 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 Lee^1,2, Danielle N. Burner^1,2, Theresa R. Mendoza^1,2, Michelle T. Muldong^1,2, Catalina Arreola^1,2, Christina N. Wu^2,3, Nicholas A. Cacalano⁴, Anna A. Kulidjian^2,5, Christopher J. Kane^1,2, Christina A. M. Jamieson^{1,2 1}Department 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 Wu^{1 1}Molecular 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. Goldstein^{2,3,4,5,6 1}Molecular 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 Vezzani^1,2, Mario Gomez-Salazar¹, Joan Casamitjana¹, Carlo Tremolada³, Bruno Péault^{1,4 1}MRC 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. Khakh^{1,2 1}Department 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. Marks^{1 1}Department 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 Padua^{1 1}Vatche 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. Liem^1,2,4, Yu Shi⁵, Xuan Wang⁵, Jiaming Shen⁵, Wei Wang^1,6,7, JiaWei Han⁵, Peipei Ping^{1,2,4,6 1}The 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 Kraut^1,3, Kevin Francis⁴, Victoria Smutko¹, Patrick Frost^{1,3 1}Greater 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 Aguilar^1,3,4, Hideaki Tsutsui^1,4, Marko Princevac¹, Monica M. Wilhelmus¹, Masaru P. Rao^{1,3,4 1}Department 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 Collins^2,4, Christopher Drake¹, Roger Slavik^2,3, R. Michael van Dam^{2,4 1}SOFIE, 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. Rajbhandari^1,2, Sarah T. Gonzalez^1,2, Michael S. Fanselow^{1,2,3 1}Department 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 Iacoboni^{2 1}Department 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 Caufield^1,2, David A. Liem^1,2,3, Anders O. Garlid^1,2, Yijiang Zhou⁴, Karol Watson^1,3, Alex A. T. Bui^1,5,6,7, Wei Wang^1,7,8,9, Peipei Ping^{1,2,3,7,8 1}The 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 Lee^1,2, Tzung K. Hsiai^{1 1}Department 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. Seidlits^{2 1}Department 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 Lee^{1 1}Department 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. Shackelford^{1 1}Division 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 Jelinek^1,2, Aimee Flores^1,3, Melanie Uebelhoer¹, Vincent Pasque², Kathrin Plath^2,3, M. Luisa Iruela-Arispe^1,3, Heather R. Christofk^2,3, William E. Lowry^1,3, Hilary A. Coller^{1,2,3 1}Department 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. Katzka^1,2, Stephanie C. Dulawa^{1 1}Department 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 Mody^1,2, Baljit S. Khakh^{1,3 1}Department 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 Touma^{1,2 1}Neonatal/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. Aldrovandi^{1 1}Department 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. Gibbs^1,2, Rachelle H. Crosbie-Watson^{1,2,3,4 1}Department 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 Lourdault^1,2, James Matsunaga^1,2, Karen V. Evangelista^1,2, David A. Haake^{1,2,3,4 1}Veterans 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. Angelovich^1,2, Anna C. Hearps^1,3, Anna Maisa¹, Theodoros Kelesidis⁴, Anthony Jaworowski^{1,3 1}Centre 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 Rossetti^{1 1}UCLA 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. Chen^1,5, Sanggu Kim^{6 1}UCLA 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. Torres^{1,2,3 1}Department 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 Matynia^{1 1}Jules 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-Arispe^{3 1}Department 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. Chen^2,3, Nusrat Khan⁴, Iain R. Murray⁴, Zaniah N. González Galofre⁴, Johnny Huard^5,6, Bruno Péault^{4,7 1}Centre 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 Kalinec^{1 1}Head 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 Arumugaswami^{1,2,3 1}Board 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 Hong^3,4, Kang Ting⁵, Wong Hee Kit^{1,4 1}Department 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 Kitchen^{1 1}David 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-Graham^{1 1}Department 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 Saghizadeh^1,2, Alexander V. Ljubimov^{1,2 1}Eye 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. Hinman^{1 1}Department 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. Cohen^{3 1}Department 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. Petrigliano^{1 1}Department 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 Allard^{1,2 1}Institute 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 Wong^{1 1}School 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. Byrne^{1,2 1}Department 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. Khakh^{1,2 1}Department 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 Banerjee^1,3, Gerald B. Call^{2 1}Department 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 Barnes^1,2,3, Nicholas C. Brecha^1,2,4, Luis Pérez De Sevilla Müller^{1 1}Department 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. Hoelzle^1,2, Bino A. Varghese^1,3, Clara K. Chan¹, Amy C. Rowat^{1 1}Department 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 Saparov^2,3, Mirko Corselli⁴, Mihaela Crisan⁵, Bo Zheng⁶, Bruno Péault^7,8, Johnny Huard^{9 1}Stem 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 Haneef^1,2, Agatha Lenartowicz³, Hsiang J. Yeh⁴, Jerome Engel Jr.⁴, John M. Stern^{4 1}Department 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. Douglas^1,2, Maureen Pisani², Rory Reid¹, Austin Head², Edward Lau², Ebrahim Mirakhor³, Jennifer Bramen², Billi Gordon², Ariana Anderson², Wesley T. Kerr², Chajoon Cheong⁴, Mark S. Cohen^{1,2 1}Neuropsychiatric 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 Yoo^{1 1}Department 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 Kamran^1,2, Konstantina-Ioanna Sereti^1,2, Peng Zhao^1,2, Shah R. Ali³, Irving L. Weissman³, Reza Ardehali^{1,2 1}Department 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 Benhaim^3,4, Patricia Zuk^{3,5 1}Cytori 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-Fuchs^1,2, Eliane Angst², Erica K. Sloan^{1,3 1}Monash 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-Kauwe^{2,3 1}Department 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. Wayne^{1 1}Department 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 Halford^1,2, Vincent Gau³, Bernard M. Churchill², David A. Haake^{2,4,5 1}Research 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. Merino^{1 1}Cardiology, 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 Ozcan^{1,2,3 1}Electrical 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. Grinnell^{1 1}Department 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 Sagasti^{1 1}Department 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 Sun^{1 1}Department 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 Scoffoni^{1 1}University 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. Vondriska^1,2,3, Sarah Franklin^{1,4 1}Department 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. Vatakis^1,2,3, Gregory C. Bristol^1,2, Sohn G. Kim^1,2, Bernard Levin^1,2, Wei Liu⁴, Caius G. Radu⁴, Scott G. Kitchen^1,2,3, Jerome A. Zack^{1,2,5 1}Department 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. Grova^1,4, Min Lee⁵, Derrick C. Wan¹, Michael T. Longaker^{1 1}Department 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 Pothoulakis^{1 1}Center 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. Gomperts^{1 1}Department 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. Kelly^{1,2 1}Department 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 Ozcan^{1,2,3 1}Electrical 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. Bayer^1,2, Yan Q. Xiong^{1,2 1}Department 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. Sanders^{1 1}Microbiology, 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 Péault^2,4, Chia Soo^{2 1}Dental 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. Lorenz^{1 1}Microbiology, 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. Sanders^{1 1}Microbiology, 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 Kim^{1 1}Department 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 Abramson^{2 1}UCLA-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. Mahajan^1,2, Jessica M. Skeie^1,2, Amir H. Assefnia^2,3, MaryAnn Mahajan^1,2, Stephen H. Tsang^{2,4 1}Department 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. Crooks^{3 1}Department 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 Ozcan^{1 1}Department 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 Pinne^1,2, David Haake^{3,4 1}Department 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 Lin^1,2,3, Ming-wei Wang⁴, Kan Liu⁵, Clifton Kwang-Fu Shen^{1,2,3 1}Department 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 Paz^{1 1}Department 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 Xu^1,2, Lin Gou^3,4, Hong-Wei Dong^{3 1}Department 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. Wilson^{6,7 1}Interdisciplinary 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 Andrews^{3,4,5,6 1}Department 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 Bitan^{1,2,3 1}Department 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 Ozcan^{1,2 1}Electrical 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 Vergara^{1 1}Department 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. Martin^{1,2,3 1}Dept. 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. Khakh^{1 1}Department 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. Khakh^{1 1}Departments 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 Sagasti^{1 1}Department 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. Duistermars^{1 1}Department 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 Bitan^{2,3 1}Department 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. Frye^{1 1}Department 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. Mikkola^{1 1}Jonsson 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 Teitell^{1 1}David 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 Teitell^{1 1}David 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 Teitell^{1 1}David 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 Teitell^{1 1}David 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-Cailliau^{1 1}Department 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-Cailliau^{1 1}Department 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-Cailliau^{1 1}Department 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 Taylor^{1 1}Department 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.