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David Geffen School of Medicine at UCLA

23 ARTICLES PUBLISHED IN JoVE

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Biology

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels
Harpreet Singh 1, Sarah Warburton 2, Thomas M. Vondriska 3, Baljit S. Khakh 1
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.

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Biology

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures
Yali Zhao 1, Dan O. Wang 1, Kelsey C. Martin 1,2,3
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.

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Biology

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

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

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Biology

Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea
Ahmed E. Hegab 1, Vi Luan Ha 1, Yasser S. Attiga 1, Derek W. Nickerson 1, Brigitte N. Gomperts 1
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.

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Immunology and Infection

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

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

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Neuroscience

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish
Rachel Einarsson 1, Marshall Haden 1, Gabrielle DiCiolli 1, Andrea Lim 1, Kolina Mah-Ginn 1, Kathleen Aguilar 1, Lucy Yazejian 1, Bruce Yazejian 1
1Natural Science Division, Pepperdine University

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

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Medicine

Quantitative Analysis of Chromatin Proteomes in Disease
Emma Monte 1, Haodong Chen 1, Maria Kolmakova 1, Michelle Parvatiyar 1, Thomas M. Vondriska 1,2,3, Sarah Franklin 1,4
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.

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Neuroscience

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures
Bruce Yazejian 1, Rita M. Yazejian 1, Rachel Einarsson 2, Alan D. Grinnell 1
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.

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Neuroscience

Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish
Yali Zhao 1, Nancy L. Wayne 1
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.

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Biology

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates
Min Zhu 1, Sepideh Heydarkhan-Hagvall 2, Marc Hedrick 1, Prosper Benhaim 3,4, Patricia Zuk 3,5
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.

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Immunology and Infection

A Protocol for Analyzing Hepatitis C Virus Replication
Songyang Ren *1, Deisy Contreras *1, Vaithilingaraja Arumugaswami 1,2
1Liver Program at Regenerative Medicine Institute, Department of Biomedical Sciences, Department of Surgery, Cedars-Sinai Medical Center, 2Department of Surgery, David Geffen School of Medicine at UCLA

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

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Medicine

Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer
Ahmed E. Hegab 1, Naofumi Kameyama 1, Aoi Kuroda 1, Shizuko Kagawa 1, Yongjun Yin 2, David Ornitz 2, Tomoko Betsuyaku 1
1Division of Pulmonary Medicine, Keio University School of Medicine, 2Department of Developmental Biology, Washington University School of Medicine

We describe a method for the detection of tumor nodule development in the lungs of an adenocarcinoma mouse model using micro-computed tomography and its use for monitoring changes in nodule size over time and in response to treatment. The accuracy of the assessment was confirmed with end-point histological quantification.

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Biochemistry

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

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

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Immunology and Infection

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons
Deisy Contreras 1, Vaithilingaraja Arumugaswami 1,2,3
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.

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Medicine

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice
Minju Song 1, Sol Kim 1, Terresa Kim 1, Sil Park 1, Ki-Hyuk Shin 1,2, Mo Kang 1,2, No-Hee Park 1,2,3, Reuben Kim 1,2
1The Shapiro Family Laboratory of Viral Oncology and Aging Research, The UCLA School of Dentistry, 2UCLA Jonsson Comprehensive Cancer Center, 3David Geffen School of Medicine at UCLA

We describe a step-by-step method of performing direct pulp capping on mice teeth for the evaluation of pulpal wound healing and reparative dentin formation in vivo.

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Bioengineering

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

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

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Immunology and Infection

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

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

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Biology

In Vivo Surface Electrocardiography for Adult Zebrafish
Yali Zhao 1, Morgan Yun 1, Sean A. Nguyen 1, Michelle Tran 1, Thao P. Nguyen 1
1Department of Medicine, Division of Cardiology, the Cardiovascular Research Laboratory, David Geffen School of Medicine at UCLA

Here, we present a reliable, minimally invasive, and cost-effective method to record and interpret electrocardiograms in live anesthetized adult zebrafish.

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Biochemistry

A Robust Method for Packing High Resolution C18 RP-nano-HPLC Columns
Yasaman Jami-Alahmadi *1, Vijaya Pandey *1, Adarsh K. Mayank *1, James A. Wohlschlegel 1
1Department of Biological Chemistry, David Geffen School of Medicine at UCLA

Here, we present and evaluate a protocol for making low cost reversed phase nano-flow liquid chromatography columns for peptide characterization using LC-MS/MS proteomic workflows.

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Biology

Determining Basal Energy Expenditure and the Capacity of Thermogenic Adipocytes to Expend Energy in Obese Mice
Michael Shum 1, Zhiqiang Zhou 2, Marc Liesa 2,3,4
1Department of Molecular Medicine, Faculty of Medicine, Universite Laval, 2Department of Medicine, Division of Endocrinology, David Geffen School of Medicine at UCLA, 3Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, 4Molecular Biology Institute at UCLA

This manuscript describes a protocol to measure the basal metabolic rate and the oxidative capacity of thermogenic adipocytes in obese mice.

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Biochemistry

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

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

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Biology

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

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

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Biology

Pipeline for Multi-Scale Three-Dimensional Anatomic Study of the Human Heart
Peter Hanna *1,2, Shumpei Mori *1, Takanori Sato 1, Shili Xu 3,4,5
1University of California Los Angeles (UCLA) Cardiac Arrhythmia Center, UCLA Health System, David Geffen School of Medicine at UCLA, 2Neurocardiology Program of Excellence, UCLA, 3Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, 4Crump Institute for Molecular Imaging, David Geffen School of Medicine at UCLA, 5Jonsson Comprehensive Cancer Center, David Geffen School of Medicine at UCLA

This protocol presents a comprehensive pipeline to analyze samples obtained from human hearts that span the microscopic and macroscopic scales.

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