University of California, Irvine View Institution's Website 95 articles published in JoVE Cancer Research Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research Stephanie J. Hachey1, Daniela Gaebler1, Christopher C. W. Hughes1,2 1Molecular Biology and Biochemistry, University of California, Irvine, 2Biomedical Engineering, University of California, Irvine This protocol presents a physiologically relevant tumor-on-a-chip model to perform high-throughput basic and translational human cancer research, advancing drug screening, disease modeling, and personalized medicine approaches with a description of loading, maintenance, and evaluation procedures. Bioengineering Light-Induced Dielectrophoresis for Characterizing the Electrical Behavior of Human Mesenchymal Stem Cells Kiara L. Lacy1, Samuel Salib1, Mary Tran2, Tunglin Tsai1, Rominna Valentine1, Herdeline Ann M. Ardoña1,2,3,4, Tayloria N. G. Adams1,2,4 1Department of Chemical and Biomolecular Engineering, Samueli School of Engineering, University of California, Irvine, 2Department of Biomedical Engineering, Samueli School of Engineering, University of California, Irvine, 3Department of Chemistry, School of Physical Sciences, University of California, Irvine, 4Sue & Bill Gross Stem Cell Research Center, University of California, Irvine Here, we present light-induced dielectrophoresis as a label-free approach for characterizing heterogeneous cell lines, specifically human mesenchymal stem cells (hMSCs). This paper describes a protocol for using and optimizing a microfluidic device with a photoconductive layer to characterize the electrical behavior of hMSCs without altering their native state. Genetics Detection of Horizontal Gene Transfer Mediated by Natural Conjugative Plasmids in E. coli Luis Mota-Bravo1, Manel Camps2, Iván Muñoz-Gutiérrez1, Andrey Tatarenkov1, Caison Warner2, Isabel Suarez1, Gerardo Cortés-Cortés2 1School of Biological Sciences, University of California Irvine, 2Department of Microbiology & Environmental Toxicology, University of California Santa Cruz Conjugation mediates horizontal gene transfer by mobilizing plasmid DNA across two different cells, facilitating the spread of beneficial genes. This work describes a widely-used method for the efficient detection of conjugative plasmid transfer, based on the use of differential markers in the conjugative plasmid, donor, and recipient to detect transconjugation. Biology Label-Retention Expansion Microscopy (LR-ExM) Enables Super-Resolution Imaging and High-Efficiency Labeling Sohyeon Park1, Yinyin Zhuang2, Xiaoyu Shi1,2,3 1Center for Complex Biological Systems, University of California Irvine, 2Department of Developmental and Cell Biology, University of California Irvine, 3Department of Chemistry, University of California Irvine A protocol of label retention expansion microscopy (LR-ExM) is demonstrated. LR-ExM uses a novel set of trifunctional anchors, which provides better labeling efficiency compared to previously introduced expansion microscopies. Neuroscience Head Implants for the Neuroimaging of Awake, Head-Fixed Rats Mehwish Bhatti1, Hayden Malone1, Gabriel Hui1, Ron D. Frostig1,2,3 1Department of Neurobiology and Behavior, School of Biological Sciences, University of California Irvine, 2Department of Biomedical Engineering, School of Engineering, University of California, Irvine, 33Center for Neurobiology of Learning and Memory, University of California, Irvine A detailed new procedure for functional imaging of awake, head-fixed rats is described. Neuroscience Full- versus Sub-Regional Quantification of Amyloid-Beta Load on Mouse Brain Sections Yuu Ohno1, Riley Murphy2, Matthew Choi3, Weijun Ou4, Rachita K. Sumbria4,5 1Henry E. Riggs School of Applied Life Sciences, Keck Graduate Institute, 2Crean College of Health and Behavioral Sciences, Chapman University, 3Keck Science Department, Claremont McKenna College, 4Department of Biomedical and Pharmaceutical Sciences, School of Pharmacy, Chapman University, 5Department of Neurology, University of California, Irvine The present protocol describes and compares the procedure to perform a full-region or sub-region of interest analysis of sagittal mouse brain sections to quantify amyloid-beta load in the APP/PS1 transgenic mouse model of Alzheimer's disease. Biology Structure-Based Simulation and Sampling of Transcription Factor Protein Movements along DNA from Atomic-Scale Stepping to Coarse-Grained Diffusion Chao E*1, Liqiang Dai*1,2, Jiaqi Tian3,4, Lin-Tai Da4, Jin Yu5,6,7 1Beijing Computational Science Research Center, 2Shenzhen JL Computational Science and Applied Research Institute, 3School of Medical Informatics and Engineering, Xuzhou Medical University, 4Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, 5Department of Physics and Astronomy, University of California, Irvine, 6Department of Chemistry, University of California, Irvine, 7NSF-Simons Center for Multiscale Cell Fate Research, University of California, Irvine The goal of this protocol is to reveal structural dynamics of one-dimensional diffusion of protein along DNA, using a plant transcription factor WRKY domain protein as an exemplary system. To do this, both atomistic and coarse-grained molecular dynamics simulations along with extensive computational samplings have been implemented. Biology A Bioinformatics Pipeline for Investigating Molecular Evolution and Gene Expression using RNA-seq Aide Macias-Muñoz1, Ali Mortazavi1 1Department of Developmental and Cell Biology, University of California, Irvine The purpose of this protocol is to investigate the evolution and expression of candidate genes using RNA sequencing data. Bioengineering Building a Simple and Versatile Illumination System for Optogenetic Experiments Phillip Kyriakakis1, Lourdes Fernandez de Cossio2, Patrick Wade Howard1, Sivleng Kouv1, Marianne Catanho1, Vincent J. Hu3, Robert Kyriakakis1, Molly E. Allen1, Yunhan Ma4, Marcelo Aguilar-Rivera1, Todd P. Coleman1 1Department of Bioengineering, University of California, San Diego, 2University of California, San Diego, 3Department of Mathematical Computational, and Systems biology, University of California, Irvine, 4Department of Biomedical Engineering, Duke University This protocol describes how to perform optogenetic experiments for controlling gene expression with red and far-red light using PhyB and PIF3. Included are step-by-step instructions for building a simple and flexible illumination system, which enables the control of gene expression or other optogenetics with a computer. Biology Time-lapse Imaging of Bacterial Swarms and the Collective Stress Response Jean-Louis Bru1, Albert Siryaporn1,2, Nina Molin Høyland-Kroghsbo3 1Department of Molecular Biology & Biochemistry, University of California, Irvine, 2Department of Physics & Astronomy, University of California, Irvine, 3Department of Veterinary and Animal Sciences, University of Copenhagen We detail a simple method to produce high-resolution time-lapse movies of Pseudomonas aeruginosa swarms that respond to bacteriophage (phage) and antibiotic stress using a flatbed document scanner. This procedure is a fast and simple method for monitoring swarming dynamics and may be adapted to study the motility and growth of other bacterial species. Bioengineering Databases to Efficiently Manage Medium Sized, Low Velocity, Multidimensional Data in Tissue Engineering Alexander R. Ochs1,2, Mehrsa Mehrabi1,2, Danielle Becker1,2, Mira N. Asad1,2, Jing Zhao1,2, Michael V. Zaragoza3,4, Anna Grosberg1,2,5,6,7 1Department of Biomedical Engineering, University of California, Irvine, 2The Edwards Lifesciences Center for Advanced Cardiovascular Technology, University of California, Irvine, 3Pediatrics-Genetics & Genomics Division-School of Medicine, University of California, Irvine, 4Biological Chemistry-School of Medicine, University of California, Irvine, 5Department of Chemical and Biomolecular Engineering, University of California, Irvine, 6Center for Complex Biological Systems, University of California, Irvine, 7The NSF-Simons Center for Multiscale Cell Fate Research (CMCF), University of California, Irvine Many researchers generate "medium-sized", low-velocity, and multi-dimensional data, which can be managed more efficiently with databases rather than spreadsheets. Here we provide a conceptual overview of databases including visualizing multi-dimensional data, linking tables in relational database structures, mapping semi-automated data pipelines, and using the database to elucidate data meaning. Neuroscience Microinjectrode System for Combined Drug Infusion and Electrophysiology M. Isabel Vanegas1, Kenneth R. Hubbard1,2, Rahim Esfandyarpour3,4, Behrad Noudoost1 1Department of Ophthalmology and Visual Sciences, University of Utah, 2Department of Biomedical Engineering, University of Utah, 3Department of Electrical Engineering and Computer Science, University of California, Irvine, 4Department of Biomedical Engineering, University of California, Irvine We present a microinjectrode system designed for electrophysiology and assisted delivery of experimental probes (i.e., nanosensors, microelectrodes), with optional drug infusion. Widely available microfluidic components are coupled to a cannula containing the probe. A step-by-step protocol for microinjectrode construction is included, with results during muscimol infusion in macaque cortex. Immunology and Infection Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes Saahir Khan1, Aarti Jain2, Omid Taghavian2, Rie Nakajima2, Algis Jasinskas2, Medalyn Supnet2, Jiin Felgner2, Jenny Davies2, Rafael Ramiro de Assis2, Sharon Jan2, Joshua Obiero2, Erwin Strahsburger2, Egest J. Pone2, Li Liang2, D. Huw Davies2, Philip L. Felgner2 1Division of Infectious Diseases, Department of Medicine, University of California Irvine Health, 2Vaccine Research and Development Center, Department of Physiology, University of California Irvine We present a protocol for using a protein microarray constructed by printing influenza antigens onto nitrocellulose-coated slides to simultaneously probe serum for multiple antibody isotypes against over 250 antigens from different virus strains, thus allowing measurement of the breadth of serum antibodies across virus subtypes. Biology Assessment of Zebrafish Lens Nucleus Localization and Sutural Integrity Irene Vorontsova1,2, James E. Hall1, Thomas F. Schilling2 1Department of Physiology and Biophysics, University of California, Irvine, 2Department of Developmental and Cell Biology, University of California, Irvine These protocols were developed to analyze cortical lens morphology, structural integrity of the zebrafish lens sutures in fixed and live lenses and to measure the position of the zebrafish lens nucleus along the anterior-posterior axis. Biochemistry Fractionation for Resolution of Soluble and Insoluble Huntingtin Species Joseph Ochaba1,2, Eva L. Morozko2,3, Jacqueline G. O’Rourke4, Leslie M. Thompson1,2,3 1Department of Psychiatry and Human Behavior, University of California Irvine, 2UCI MIND, University of California Irvine, 3Department of Neurobiology and Behavior, University of California Irvine, 4Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center A method is described for fractionation of insoluble and soluble mutant huntingtin species from mouse brain and cell culture. The method described is useful for characterization and quantification of huntingtin protein flux and aids in analyzing protein homeostasis in disease pathogenesis and in the presence of perturbations Genetics Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage Xiangduo Kong*1, Gladys M.S. Cruz*2, Bárbara A. Silva2, Nicole M. Wakida2, Nima Khatibzadeh2, Michael W. Berns2,3,4, Kyoko Yokomori1 1Department of Biological Chemistry, School of Medicine, University of California, Irvine, 2Beckman Laser Institute and Medical Clinic, University of California, Irvine, 3Department of Developmental and Cell Biology, School of Biological Sciences, University of California, Irvine, 4Department of Biomedical Engineering and Surgery, University of California, Irvine The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo. Engineering Fabrication of 3D Carbon Microelectromechanical Systems (C-MEMS) Bidhan Pramanick1, Sergio O. Martinez-Chapa1, Marc Madou1,2, Hyundoo Hwang1,3 1School of Engineering and Sciences, Tecnologico de Monterrey, 2Department of Mechanical and Aerospace Engineering, University of California, 3BBB Inc Long and hollow glassy carbon microfibers were fabricated based on the pyrolysis of a natural product, human hair. The two fabrication steps of carbon microelectromechanical and carbon nanoelectromechanical systems, or C-MEMS and C-NEMS, are: (i) photolithography of a carbon-rich polymer precursor and (ii) pyrolysis of the patterned polymer precursor. Cancer Research Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm Thanh Kim Nguyen1, Sarah J. Morse1, Angela G. Fleischman1 1Department of Medicine, Division of Hematology/Oncology, University of California, Irvine This manuscript provides a description of the methodology used to establish transduction-transplantation mouse models. A detailed account is given of technical errors to avoid when performing bone marrow transplants. A clear understanding should be gained of the importance of high viral titer, transfection/transduction, and irradiation. Biochemistry Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase Elisa Romeo1, Silvia Pontis1, Stefano Ponzano1, Fabiola Bonezzi1, Marco Migliore1, Simona Di Martino1, Maria Summa1, Daniele Piomelli1,2 1Drug Discovery and Development, Istituto Italiano di Tecnologia, 2Departments of Anatomy and Neurobiology, Pharmacology, and Biological Chemistry, University of California, Irvine School of Medicine Here, we describe the preparation and use of an activity-based probe (ARN14686, undec-10-ynyl-N-[(3S)-2-oxoazetidin-3-yl]carbamate) that allows for the detection and quantification of the active form of the proinflammatory enzyme N-acylethanolamine acid amidase (NAAA), both in vitro and ex vivo. Neuroscience Determination of Photoreceptor Cell Spectral Sensitivity in an Insect Model from In Vivo Intracellular Recordings Kyle J. McCulloch1, Daniel Osorio2, Adriana D. Briscoe1 1Department of Ecology and Evolutionary Biology, University of California, Irvine, 2School of Life Sciences, University of Sussex The electrophysiological technique of intracellular recording is demonstrated and used to determine spectral sensitivities of single photoreceptor cells in the compound eye of a butterfly. Biology Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis Edmund Ong1,2, Catherine Cahill1,2,3 1Department of Anesthesiology and Perioperative Care, University of California Irvine, 2 Receptor trafficking modulates signaling and cell responsiveness to ligands and is, itself, responsive to cell conditions, including ligand-induced signaling. Here, we describe a powerful and flexible technique for quantitatively assessing drug-induced receptor trafficking using immunolabeling and colocalizational analysis. Biology Imaging Local Ca2+ Signals in Cultured Mammalian Cells Jeffrey T. Lock1, Kyle L. Ellefsen1, Bret Settle1, Ian Parker1,2, Ian F. Smith1 1Neurobiology and Behavior, University of California, Irvine, 2Physiology and Biophysics, University of California, Irvine Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events. Neuroscience Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord Jason G. Weinger1, Milton L. Greenberg2, Melanie P. Matheu4, Ian Parker3, Craig M. Walsh1, Thomas E. Lane5, Michael D. Cahalan2 1Molecular Biology and Biochemistry, University of California, Irvine, 2Physiology and Biophysics, University of California, Irvine, 3Neurobiology and Behavior, University of California, Irvine, 4University of California San Francisco Diabetes Center, University of California, San Francisco, 5Pathology, University of Utah A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord. Bioengineering From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope Carmine Di Rienzo1,2, Enrico Gratton3, Fabio Beltram1, Francesco Cardarelli2 1NEST Laboratory, Scuola Normale Superiore, 2Center for Nanotechnology Innovation, Instituto Italiano di Tecnologia, 3Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups. Bioengineering 3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles Andrea Anzalone*1, Paolo Annibale*1, Enrico Gratton1 1Biomedical Engineering, Laboratory for Fluorescence Dynamics, University of California, Irvine In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope. Biology Generation of Transgenic Hydra by Embryo Microinjection Celina E. Juliano1, Haifan Lin1, Robert E. Steele2 1Yale Stem Cell Center and Department of Cell Biology, Yale University School of Medicine, 2Department of Biological Chemistry and the Developmental Biology Center, University of California, Irvine Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi. Immunology and Infection High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages Jing Wu1, Roberta Pugh1, Richard C. Laughlin1, Helene Andrews-Polymenis2, Michael McClelland3, Andreas J. Bäumler4, L. Garry Adams1 1Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, 2Department of Microbial and Molecular Pathogenesis, College of Medicine, Texas A&M University System Health Science Center, 3University of California, Irvine, 4Department of Medical Microbiology & Immunology, School of Medicine, University of California, Davis A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions. Engineering Process of Making Three-dimensional Microstructures using Vaporization of a Sacrificial Component Du T. Nguyen1, Y. T. Leho2, Aaron P. Esser-Kahn2 1Department of Physics, University of California, Irvine, 2Department of Chemistry, University of California, Irvine The Vaporization of a Sacrificial Component (VaSC) process is used to fabricate microvascular structures. This procedure uses sacrificial poly(lactic) acid fibers to form hollow microchannels with precise 3D geometric positioning provided by laser micromachined guide plates. Biology Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue Hassan Khalil1, Wenxian Nie1, Robert A Edwards2, James Yoo1 1Department of Surgery, UCLA, 2Department of Pathology, UC Irvine The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation. Biology Assessing Differences in Sperm Competitive Ability in Drosophila Shu-Dan Yeh1, Carolus Chan1, José M. Ranz1 1Department of Ecology and Evolutionary Biology, University of California, Irvine Differential sperm competitive ability among Drosophila males with distinct genotypes can be ascertained through double-mating experiments. Each of these experiments involves one of the males of interest and a reference male. Readily identifiable markers in the progeny allow inference of the fraction of individuals fathered by each male. Behavior Creating Dynamic Images of Short-lived Dopamine Fluctuations with lp-ntPET: Dopamine Movies of Cigarette Smoking Evan D. Morris1,2,3,4, Su Jin Kim1,3, Jenna M. Sullivan1,3,4, Shuo Wang3,4, Marc D. Normandin5, Cristian C. Constantinescu6, Kelly P. Cosgrove1,2,3 1Diagnostic Radiology, Yale University, 2Psychiatry, Yale University, 3Yale PET Center, Yale University, 4Biomedical Engineering, Yale University, 5Nuclear Medicine, Massachusetts General Hospital, 6Radiological Sciences, University of California, Irvine We present a novel PET imaging approach for capturing dopamine fluctuations induced by cigarette smoking. Subjects smoke in the PET scanner. Dynamic PET images are modeled voxel-by-voxel in time by lp-ntPET, which includes a time-varying dopamine term. The results are 'movies' of dopamine fluctuations in the striatum during smoking. Medicine Permanent Cerebral Vessel Occlusion via Double Ligature and Transection Melissa F. Davis*1,2, Christopher Lay*1,2,3, Ron D. Frostig1,2,3,4 1Department of Neurobiology & Behavior, University of California, Irvine, 2The Center for the Neurobiology of Learning and Memory, University of California, Irvine, 3The Center for Hearing Research, University of California, Irvine, 4Department of Biomedical Engineering, University of California, Irvine We describe a highly reproducible method for the permanent occlusion of a rodent major cerebral blood vessel. This technique can be accomplished with very little peripheral damage, minimal blood loss, a high rate of long-term survival, and consistent infarct volume commensurate with the human clinical population. Medicine The Goeckerman Regimen for the Treatment of Moderate to Severe Psoriasis Rishu Gupta1,2, Maya Debbaneh2,3, Daniel Butler2,4, Monica Huynh2,5, Ethan Levin2, Argentina Leon2, John Koo2, Wilson Liao2 1Keck School of Medicine, University of Southern California, 2Psoriasis and Skin Treatment Center Dermatology, University of California, San Francisco, 3University of California Irvine School of Medicine, 4University of Arizona College of Medicine, 5Chicago College of Osteopathic Medicine Psoriasis is a chronic, immune-mediated inflammatory skin disease. The Goeckerman regimen, formulated for the treatment of psoriasis, consists of exposure to ultraviolet B (UVB) light and application of crude coal tar (CCT). The following protocol is for the administration of Goeckerman therapy for the treatment of moderate-to-severe psoriasis. Neuroscience Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve Michelle R. Allen-Sharpley1, Michelle Tjia1, Karina S. Cramer1 1Department of Neurobiology and Behavior, University of California, Irvine Here we describe a microdissection technique followed by fluorescent dye injection into the acoustic ganglion of early chick embryos for selective tracing of auditory axon fibers in the nerve and hindbrain. Medicine Generation of Alginate Microspheres for Biomedical Applications Omaditya Khanna1, Jeffery C. Larson2, Monica L. Moya3, Emmanuel C. Opara4, Eric M. Brey2,5 1Department of Chemical and Biological Engineering, Illinois Institute of Technology, 2Department of Biomedical Engineering, Illinois Institute of Technology, 3Department of Biomedical Engineering, University of California at Irvine, 4Wake Forest Institute for Regenerative Medicine and Department of Biomedical Engineering, Wake Forest University Health Sciences, 5Research Service, Hines Veterans Administration Hospital In the following sections, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. We specifically illustrate a technique for creating multilayered alginate microspheres for the dual purpose of cell and protein encapsulation as a potential treatment for type 1 diabetes. Immunology and Infection Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes Jennifer Juhn1, Anthony A. James1,2 1Department of Molecular Biology and Biochemistry, University of California, Irvine, 2Department of Microbiology and Molecular Genetics, University of California, Irvine Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes. Biology Isolation and Purification of Kinesin from Drosophila Embryos Robilyn Sigua1, Suvranta Tripathy1, Preetha Anand1, Steven P. Gross1 1Department of Developmental and Cell Biology, School of Biosciences, University of California, Irvine This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition. Biology Quantifying Agonist Activity at G Protein-coupled Receptors Frederick J. Ehlert1, Hinako Suga2, Michael T. Griffin3 1Department of Pharmacology, University of California, Irvine, 2Department of Pharmacology, University of California, 3Schmid College of Science, Chapman University A method for estimating the affinity constant of an agonist for the active state (Kb) of a G protein-coupled receptor is described. The analysis provides absolute or relative measures of Kb depending on whether constitutive receptor activation is measurable. Our method applies to various responses downstream from receptor activation. Medicine Quantifying Cognitive Decrements Caused by Cranial Radiotherapy Lori- Ann Christie1, Munjal M. Acharya1, Charles L. Limoli1 1Department of Radiation Oncology, University of California Irvine Cognitive impairment resulting from the radiotherapeutic management of brain tumors represents a clinically intractable condition that adversely impacts quality of life. The capability to critically evaluate potential interventions for ameliorating radiation-induced cognitive decrements ultimately depends on the capability to undertake rigorous quantitative assessments of cognition. Medicine Stem Cell Transplantation Strategies for the Restoration of Cognitive Dysfunction Caused by Cranial Radiotherapy Munjal M. Acharya*1, Dante E. Roa*1, Omar Bosch1, Mary L. Lan*1, Charles L. Limoli1 1Department of Radiation Oncology, University of California Irvine Brain tumor patients routinely undergo cranial radiotherapy, and while beneficial, this treatment often results in debilitating cognitive dysfunction. This serious unresolved problem has at present, no clinical recourse, and has driven our efforts to devise stem cell based therapies for the recovery of radiation-induced cognitive decrements. Neuroscience Mapping Inhibitory Neuronal Circuits by Laser Scanning Photostimulation Taruna Ikrar1, Nicholas D. Olivas1, Yulin Shi1, Xiangmin Xu1,2 1Department of Anatomy and Neurobiology, School of Medicine, University of California, Irvine, 2Department of Biomedical Engineering, School of Engineering, University of California, Irvine This paper introduces an approach of combining laser scanning photostimulation with whole cell recordings in transgenic mice expressing GFP in limited inhibitory neuron populations. The technique allows for extensive mapping and quantitative analysis of local synaptic circuits of specific inhibitory cortical neurons. Neuroscience Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus Kevin S. Carbajal1,2, Jason G. Weinger1,2, Lucia M. Whitman1,2, Chris S. Schaumburg1,2, Thomas E. Lane1,2,3 1Department of Molecular Biology and Biochemistry, University of California, Irvine, 2Sue and Bill Gross Stem Cell Center, University of California, Irvine, 3Institute for Immunology, University of California, Irvine The transplantation of mouse neural stem cells (NSCs) into the spinal cords of mice with established demyelination is detailed. The preparation of NSCs, the laminectomy of thoracic vertebra 9 (T9), and transplantation of NSCs is outlined along with the pre- and post-operative care of the mice. Neuroscience Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures Ardeshir S. Rahman1, Shaudee Parvinjah1, Michael A. Hanna1, Pablo R. Helguera1, Jorge Busciglio1 1Department of Neurobiology and Behavior, University of California, Irvine Here, we describe a method for efficient cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. This simple protocol provides flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures. Biology Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH Melanie P. Matheu1, Christine Beeton1, Ian Parker2, K. George Chandy1, Michael D. Cahalan1 1Department of Physiology and Biophysics, University of California, Irvine (UCI), 2Department of Neurobiology and Behavior, University of California, Irvine (UCI) Here we demonstrate a method for inducing and recording the progress of a delayed type-hypersensitivity (DTH) reaction in the rat ear. This is followed by a demonstration of the preparation of rat ear tissue for two-photon imaging of the effector / memory T cell response. Biology Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging Melanie P. Matheu1, Debasish Sen1, Michael D Cahalan1, Ian Parker2 1Department of Physiology and Biophysics, University of California, Irvine (UCI), 2Department of Neurobiology and Behaviour, University of California, Irvine (UCI) Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging. Biology Rapid Genotyping of Mouse Tissue Using Sigma's Extract-N-Amp Tissue PCR Kit Linda Doan1, Edwin S. Monuki1 1Department of Developmental and Cell Biology, University of California, Irvine (UCI) The complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, is done in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR kit. Biology Murine Skin Transplantation Kym R. Garrod1, Michael D. Cahalan1 1Department of Physiology and Biophysics, University of California, Irvine (UCI) Allogeneic skin transplantation is a standard model to assay host T cell responses to MHC-disparate donor antigens. This video-article provides a visual tutorial of each step involved in performing a BALB/c-->C57BL/6 skin transplant. Biology Preparation of Dissociated Mouse Cortical Neuron Cultures Lutz G. W. Hilgenberg1, Martin A. Smith1 1Department of Anatomy and Neurobiology, University of California, Irvine (UCI) This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation. Biology Isolation of Mononuclear Cells from the Central Nervous System of Rats with EAE Christine Beeton1, K. George Chandy1 1Department of Physiology and Biophysics, University of California, Irvine (UCI) In this video we demonstrate how to isolate mononuclear cells from the central nervous system of rats with experimental autoimmune encephalomyelitis. Biology BioMEMS: Forging New Collaborations Between Biologists and Engineers Noo Li Jeon1 1Department of Biomedical Engineering, University of California, Irvine (UCI) Biology Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices Joseph Harris1, Hyuna Lee1, Behrad Vahidi1, Cristina Tu2, David Cribbs3, Carl Cotman3, Noo Li Jeon1 1Department of Biomedical Engineering, University of California, Irvine (UCI), 2Stem Cell Research Center, University of California, Irvine (UCI), 3Institute for Brain Aging and Dementia, University of California, Irvine (UCI) In this video we demonstrate how to use the neuron microfluidic device without plasma bonding. Biology Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification Melanie P. Matheu1, Michael D. Cahalan1 1Department of Physiology and Biophysics, University of California, Irvine (UCI) Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically ≥ 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi. Biology RNA Extraction from Neuroprecursor Cells Using the Bio-Rad Total RNA Kit Jia Sheng Su1, Edwin S. Monuki2 1Department of Developmental and Cell Biology, University of California, Irvine (UCI), 2Department of Pathology, University of California, Irvine (UCI) Biology Preparing T Cell Growth Factor from Rat Splenocytes Christine Beeton1, K. George Chandy1 1Department of Physiology and Biophysics, University of California, Irvine (UCI) We describe the preparation of T cell growth factor used for the in vitro expansion of antigen-specific rat T lymphocyte lines. Biology Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device Joseph Harris1, Hyuna Lee1, Christina Tu Tu2, David Cribbs3, Carl Cotman3, Noo Li Jeon1 1Department of Biomedical Engineering, University of California, Irvine (UCI), 2Stem Cell Research Center, University of California, Irvine (UCI), 3Institute for Brain Aging and Dementia, University of California, Irvine (UCI) In this video we demonstrate the preparation of E18 Cortical Rat Neurons. Biology Windowing Chicken Eggs for Developmental Studies Matthew J. Korn1, Karina S. Cramer1 1Department of Neurobiology and Behaviour, University of California, Irvine (UCI) The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling. In this article we demonstrate one egg preparation method that has been optimized for long survival times. Biology Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos Matthew J. Korn1, Karina S. Cramer1 1Department of Neurobiology and Behaviour, University of California, Irvine (UCI) A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos. Biology Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs Victor Chi1, K. George Chandy1 1Department of Physiology and Biophysics, University of California, Irvine (UCI) Biology Laser Capture Microdissection of Mammalian Tissue Robert A Edwards1 1Department of Pathology, University of California, Irvine (UCI) Biology Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats Christine Beeton1, K. George Chandy1 1Department of Physiology and Biophysics, University of California, Irvine (UCI) Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T (TEM) lymphocytes. Here we demonstrate how to activate antigen-specific TEM cells, induce adoptive DTH in Lewis rats and monitor the inflammatory response. Biology Enrichment of NK Cells from Human Blood with the RosetteSep Kit from StemCell Technologies Christine Beeton1, K. George Chandy1 1Department of Physiology and Biophysics, University of California, Irvine (UCI) Natural killer cells are a small population of lymphocytes. Here we show how to isolate these cells from human blood by negative selection, using a kit from StemCell Technologies. The cells obtained are viable and untouched by antibodies, and therefore ready to be used for a number of procedures. Biology Immunocytochemistry: Human Neural Stem Cells Steven Marchenko1, Lisa Flanagan1 1Department of Pathology, University of California, Irvine (UCI) Immunocytochemistry is a powerful method to determine the presence, subcellular localization, and relative abundance of an antigen of interest in cultured cells. This protocol presents an easy-to-follow series of steps that will enable one to conserve antibodies and get the most out of one's staining. Biology Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice Melanie P. Matheu1, Ian Parker2, Michael D. Cahalan1 1Department of Physiology and Biophysics, University of California, Irvine (UCI), 2Department of Neurobiology and Behaviour, University of California, Irvine (UCI) Two-photon imaging has uncovered lymphocyte motility and cellular interactions within the lymph node under basal conditions and durring an immune response 1. Here, we demonstrate adoptive transfer of T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node. Biology Drawing Blood from Rats through the Saphenous Vein and by Cardiac Puncture Christine Beeton1, Adriana Garcia1, K. George Chandy1 1Department of Physiology and Biophysics, University of California, Irvine (UCI) Blood draws are necessary in a large number of studies, for example to study the pharmacokinetics profile of a compound. Here, we demonstrate how to draw blood from rats using two techniques: blood draw from the saphenous vein or by cardiac puncture. Biology Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons Joseph Harris1, Hyuna Lee1, Behrad Vahidi1, Christina Tu2, David Cribbs3, Noo Li Jeon1, Carl Cotman3 1Department of Biomedical Engineering, University of California, Irvine (UCI), 2Stem Cell Research Center, University of California, Irvine (UCI), 3Institute for Brain Aging and Dementia, University of California, Irvine (UCI) In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons. Biology Counting Human Neural Stem Cells Steven Marchenko1, Lisa Flanagan1 1Department of Pathology, University of California, Irvine (UCI) Knowledge of the exact number of viable cells is required for many tissue culture manipulations. This protocol describes how to differentiate between live and dead cells and quantify cells using a hemacytometer. Although it describes counting human neural stem/precursor cells (hNSPCs), it can be used for other cell types. Biology Passaging Human Neural Stem Cells Steven Marchenko1, Lisa Flanagan1 1Department of Pathology, University of California, Irvine (UCI) The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell research. Biology Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels Aubin Penna1, Michael Cahalan1 1Department of Physiology and Biophysics, University of California, Irvine (UCI) This technical article describes a standard western-blotting procedure using the commercially available NuPAGE electrophoresis Mini-Gel system from Invitrogen. Biology Transformation of Plasmid DNA into E. coli Using the Heat Shock Method Alexandrine Froger1, James E. Hall1 1Department of Physiology and Biophysics, University of California, Irvine (UCI) Biology Isolation of Genomic DNA from Mouse Tails Tony Zangala1 1Department of Physiology and Biophysics, University of California, Irvine (UCI) Biology Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit Shenyuan Zhang1, Michael D. Cahalan1 1Department of Physiology and Biophysics, University of California, Irvine (UCI) Biology Whole Cell Recordings from Brain of Adult Drosophila Huaiyu Gu1, Diane K. O'Dowd1 1Department of Development and Cell Biology, Department of Anatomy and Neurobiology, University of California, Irvine (UCI) This video demonstrates the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons using standard whole cell technology. It includes images of GFP labeled cells and neurons viewed during recording. Biology Induction and Monitoring of Active Delayed Type Hypersensitivity (DTH) in Rats Christine Beeton1, K. George Chandy1 1Department of Physiology and Biophysics, University of California, Irvine (UCI) Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes. Here we demonstrate how to induce active DTH in Lewis rats and monitor the inflammatory response. Biology Transfecting Human Neural Stem Cells with the Amaxa Nucleofector Steven Marchenko1, Lisa Flanagan1 1Department of Pathology, University of California, Irvine (UCI) Introducing a gene of interest into a cell is a powerful method for elucidating its function in vivo. This protocol describes an efficient method of transfecting a culture of human neural stem/precursor cells (hNSPCs) using the Nucleofector electroporation apparatus made by Amaxa. Biology Injection of dsRNA into Female A. aegypti Mosquitos Brian M. Luna1, Jennifer Juhn1, Anthony A. James2 1Department of Molecular Biology and Biochemistry, University of California, Irvine (UCI), 2Department of Molecular Biology and Biochemistry, Department of Microbiology and Molecular Genetics, University of California, Irvine (UCI) Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the James lab to inject dsRNA into female A. aegypti mosquitoes, which harbor the dengue virus. The technique for calibrating injection needles, manipulating the injection setup, and injecting dsRNA into the thorax is illustrated. Biology Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes Olle Terenius1, Jennifer Juhn1, Anthony A. James2 1Department of Molecular Biology and Biochemistry, University of California, Irvine (UCI), 2Department of Molecular Biology and Biochemistry, Department of Microbiology and Molecular Genetics, University of California, Irvine (UCI) Anopheles stephensi mosquitoes are vectors for malaria inhabiting India and throughout Asia. This video demonstrates the technique for performing microinjections of this species with transgenes that will confer resistance to the malaria to the mosquito. Much of the methodology demonstrated in this video is applicable to microinjection techniques of other mosquito species. Biology Microinjection of A. aegypti Embryos to Obtain Transgenic Mosquitoes Nijole Jasinskiene1, Jennifer Juhn1, Anthony A. James2 1Department of Molecular Biology and Biochemistry, University of California, Irvine (UCI), 2Department of Molecular Biology and Biochemistry, Department of Microbiology and Molecular Genetics, University of California, Irvine (UCI) In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever. The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated. Biology Induction and Clinical Scoring of Chronic-Relapsing Experimental Autoimmune Encephalomyelitis Christine Beeton1, Adriana Garcia1, K. George Chandy1 1Department of Physiology and Biophysics, University of California, Irvine (UCI) This video demonstrates the induction and clinical scoring of an animal model of multiple sclerosis: chronic-relapsing experimental autoimmune encephalomyelitis in DA rats. The disease, induced by immunizing rats with an emulsion containing whole rat spinal cord and complete Freund's adjuvant, presents clinical signs resembling the human disease. Biology Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos Beatriz Sicaeros1, Diane K. O'Dowd1 1Department of Development and Cell Biology, Department of Anatomy and Neurobiology, University of California, Irvine (UCI) This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections. Biology Dissection of Midgut and Salivary Glands from Ae. aegypti Mosquitoes Judy Coleman1, Jennifer Juhn1, Anthony A. James2 1Department of Molecular Biology and Biochemistry, University of California, Irvine (UCI), 2Department of Molecular Biology and Biochemistry, Department of Microbiology and Molecular Genetics, University of California, Irvine (UCI) The mosquito midgut and salivary glands are key entry and exit points for vector pathogens like Plasmodium falciparum and the dengue virus. This video demonstrates the dissection techniques for removing the midgut and salivary glands from Aedes aegypti mosquitoes. Biology Preventing the Spread of Malaria and Dengue Fever Using Genetically Modified Mosquitoes Anthony A. James1 1Department of Molecular Biology and Biochemistry, Department of Microbiology and Molecular Genetics, University of California, Irvine (UCI) In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission. Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed. Biology Flash Freezing and Cryosectioning E12.5 Mouse Brain D. Spencer Currle1, Edwin S. Monuki1 1Department of Developmental and Cell Biology, University of California, Irvine (UCI) Demonstrated in this video are the techniques for flash freezing and sectioning embryonic brain tissue from mouse. Useful tips for using the cryostat are given, including troubleshooting methods that can be used while cutting to ensure that the resultant tissues sections are free of cracks and other distortions. Biology Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae Beatriz Sicaeros1, Jorge M. Campusano1, Diane K. O'Dowd1 1Department of Development and Cell Biology, Department of Anatomy and Neurobiology, University of California, Irvine (UCI) This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2. Biology Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis Martin N. Nakatsu1, Jaeger Davis1, Christopher C.W. Hughes1 1Department of Molecular Biology and Biochemistry, University of California, Irvine (UCI) This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown. Biology Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay Souichi Ogata1, Ken W.Y. Cho1 1Department of Developmental and Cell Biology, University of California, Irvine (UCI) This video demonstrates the technique used for preparation of organizer and animal pole explants from Xenopus laevis embryos, including the use of the eyebrow knife - a specialized dissection tool made of one's eyebrow. The protocol for assembling an adhesion assay is also given, which probes for the presence of key adhesion molecules present on the surface organizer or animal pole cells that are critical for proper development. Biology Plastic Embedding and Sectioning of Xenopus laevis Embryos Souichi Ogata1, Shimako Kawauchi1, Anne Calof2, Ken W.Y. Cho1 1Department of Developmental and Cell Biology, University of California, Irvine (UCI), 2University of California, Irvine (UCI) Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video. Biology Christopher Hughes: An in vitro model for the Study of Angiogenesis (Interview) Christopher C.W. Hughes1 1Department of Molecular Biology and Biochemistry, University of California, Irvine (UCI) Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro. He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis. Biology Isolation of Human Umbilical Vein Endothelial Cells (HUVEC) Jaeger Davis1, Steve P. Crampton1, Christopher C.W. Hughes1 1Department of Molecular Biology and Biochemistry, University of California, Irvine (UCI) This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab. Biology Harvesting Sperm and Artificial Insemination of Mice Amanda R. Duselis1, Paul B. Vrana1 1Dept. of Biological Chemistry, University of California, Irvine (UCI) This protocol demonstrates methods for extracting sperm from the testes of males and then inseminating female mice. This procedure is useful when precise time is needed in developmental studies as well as transgenic work. Biology Retrieval of Mouse Oocytes Amanda R. Duselis1, Paul B. Vrana1 1Dept. of Biological Chemistry, University of California, Irvine (UCI) This protocol illustrates the technique for extracting oocytes or early-stage fertilized embryos from the oviduct of mice. The ability to identify the infindibulum and insert a blunt end needle into it is essential to correctly performing the procedure. Biology Culture of Mouse Neural Stem Cell Precursors D. Spencer Currle1, Jia Sheng Hu2, Aaron Kolski-Andreaco3, Edwin S. Monuki2 1Department of Developmental and Cell Biology, University of California, Irvine (UCI), 2Department of Pathology, University of California, Irvine (UCI), 3Department of Physiology and Biophysics, University of California, Irvine (UCI) This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown. Biology Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae Dianne C. Purves1, Carrie Brachmann1 1Department of Developmental and Cell Biology, University of California, Irvine (UCI) This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization. Biology Growth Factor-Coated Bead Placement on Dorsal Forebrain Explants D. Spencer Currle1, Aaron Kolski-Andreaco2, Edwin S. Monuki3 1Department of Developmental and Cell Biology, University of California, Irvine (UCI), 2Department of Physiology and Biophysics, University of California, Irvine (UCI), 3Department of Pathology, University of California, Irvine (UCI) This video demonstrates two methods for preparing and placing beads, which have been coated with growth factor, on explants of the developing cerebral cortex. These beads can be used to induce spatially restricted gene expression on developing neural tissue such as forebrain explants. Methods are given for using both Affi-Gel beads and heparin acryllic beads. Biology Mouse Dorsal Forebrain Explant Isolation Spencer Currle1, Aaron Kolski-Andreaco2, Edwin S. Monuki3 1Department of Developmental and Cell Biology, University of California, Irvine (UCI), 2Department of Physiology and Biophysics, University of California, Irvine (UCI), 3Department of Pathology, University of California, Irvine (UCI) This video demonstrates the protocol for isolating and culturing explants of the mouse forebrain from embyonic day 12 mice. Procedures for removal of the uterus, embryos from uterus, and dissection of embryos are given. In addition the methodology for transferring these explants onto specialized membranes on which they are cultured is demonstrated. The development of the forebrain can be studied in vitro using this preparations as well as changes in gene expression. Biology Mouse Adrenal Chromaffin Cell Isolation Aaron Kolski-Andreaco1, Haijiang Cai2,3, D. Spencer Currle4, K. George Chandy1, Robert H. Chow2,3 1Department of Physiology and Biophysics, University of California, Irvine (UCI), 2Department of Physiology and Biophysics, University of Southern California, Keck School of Medicine, 3Zilkha Neurogenetic Institute, University of Southern California, Keck School of Medicine, 4Department of Developmental and Cell Biology, University of California, Irvine (UCI) Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting. This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex. The digestion of the medulla into single chromaffin cells is then demonstrated.
