University of California, Irvine

90 articles published in JoVE

• Biology

  
  Label-Retention Expansion Microscopy (LR-ExM) Enables Super-Resolution Imaging and High-Efficiency Labeling Sohyeon Park¹, Yinyin Zhuang², Xiaoyu Shi^{1,2,3 1}Center 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

  
  Full- versus Sub-Regional Quantification of Amyloid-Beta Load on Mouse Brain Sections Yuu Ohno¹, Riley Murphy², Matthew Choi³, Weijun Ou⁴, Rachita K. Sumbria^{4,5 1}Henry 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 Tian^3,4, Lin-Tai Da⁴, Jin Yu^{5,6,7 1}Beijing 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 Mortazavi^{1 1}Department 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.

• Biology

  
  Time-lapse Imaging of Bacterial Swarms and the Collective Stress Response Jean-Louis Bru¹, Albert Siryaporn^1,2, Nina Molin Høyland-Kroghsbo^{3 1}Department 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. Ochs^1,2, Mehrsa Mehrabi^1,2, Danielle Becker^1,2, Mira N. Asad^1,2, Jing Zhao^1,2, Michael V. Zaragoza^3,4, Anna Grosberg^{1,2,5,6,7 1}Department 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. Hubbard^1,2, Rahim Esfandyarpour^3,4, Behrad Noudoost^{1 1}Department 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. Felgner^{2 1}Division 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 Vorontsova^1,2, James E. Hall¹, Thomas F. Schilling^{2 1}Department 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 Ochaba^1,2, Eva L. Morozko^2,3, Jacqueline G. O’Rourke⁴, Leslie M. Thompson^{1,2,3 1}Department 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. Berns^2,3,4, Kyoko Yokomori^{1 1}Department 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 Madou^1,2, Hyundoo Hwang^{1,3 1}School 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. Fleischman^{1 1}Department 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 Piomelli^{1,2 1}Drug 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. Briscoe^{1 1}Department 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 Ong^1,2, Catherine Cahill^{1,2,3 1}Department 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 Parker^1,2, Ian F. Smith^{1 1}Neurobiology 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. Cahalan^{2 1}Molecular 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 Rienzo^1,2, Enrico Gratton³, Fabio Beltram¹, Francesco Cardarelli^{2 1}NEST 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 Gratton^{1 1}Biomedical 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. Steele^{2 1}Yale 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 Adams^{1 1}Department 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-Kahn^{2 1}Department 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 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.

• Biology

  
  Assessing Differences in Sperm Competitive Ability in Drosophila Shu-Dan Yeh¹, Carolus Chan¹, José M. Ranz^{1 1}Department 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. Morris^1,2,3,4, Su Jin Kim^1,3, Jenna M. Sullivan^1,3,4, Shuo Wang^3,4, Marc D. Normandin⁵, Cristian C. Constantinescu⁶, Kelly P. Cosgrove^{1,2,3 1}Diagnostic 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. Frostig^{1,2,3,4 1}Department 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 Gupta^1,2, Maya Debbaneh^2,3, Daniel Butler^2,4, Monica Huynh^2,5, Ethan Levin², Argentina Leon², John Koo², Wilson Liao^{2 1}Keck 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. Cramer^{1 1}Department 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. Brey^{2,5 1}Department 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. James^{1,2 1}Department 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. Gross^{1 1}Department 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. Griffin^{3 1}Department 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. Limoli^{1 1}Department 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. Limoli^{1 1}Department 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 Xu^{1,2 1}Department 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. Carbajal^1,2, Jason G. Weinger^1,2, Lucia M. Whitman^1,2, Chris S. Schaumburg^1,2, Thomas E. Lane^{1,2,3 1}Department 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 Busciglio^{1 1}Department 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. Cahalan^{1 1}Department 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 Parker^{2 1}Department 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. Monuki^{1 1}Department 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. Cahalan^{1 1}Department 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. Smith^{1 1}Department 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 Chandy^{1 1}Department 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 Jeon^{1 1}Department 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 Jeon^{1 1}Department 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. Cahalan^{1 1}Department 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. Monuki^{2 1}Department 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 Chandy^{1 1}Department 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 Jeon^{1 1}Department 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. Cramer^{1 1}Department 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. Cramer^{1 1}Department 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 Chandy^{1 1}Department of Physiology and Biophysics, University of California, Irvine (UCI)

• Biology

  
  Laser Capture Microdissection of Mammalian Tissue Robert A Edwards^{1 1}Department of Pathology, University of California, Irvine (UCI)

• Biology

  
  Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats Christine Beeton¹, K. George Chandy^{1 1}Department 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 Chandy^{1 1}Department 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.

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  Immunocytochemistry: Human Neural Stem Cells Steven Marchenko¹, Lisa Flanagan^{1 1}Department 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.

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  Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice Melanie P. Matheu¹, Ian Parker², Michael D. Cahalan^{1 1}Department 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.

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  Drawing Blood from Rats through the Saphenous Vein and by Cardiac Puncture Christine Beeton¹, Adriana Garcia¹, K. George Chandy^{1 1}Department 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.

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  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 Cotman^{3 1}Department 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.

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  Counting Human Neural Stem Cells Steven Marchenko¹, Lisa Flanagan^{1 1}Department 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.

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  Passaging Human Neural Stem Cells Steven Marchenko¹, Lisa Flanagan^{1 1}Department 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.

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  Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels Aubin Penna¹, Michael Cahalan^{1 1}Department 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. Hall^{1 1}Department of Physiology and Biophysics, University of California, Irvine (UCI)

• Biology

  
  Isolation of Genomic DNA from Mouse Tails Tony Zangala^{1 1}Department of Physiology and Biophysics, University of California, Irvine (UCI)

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  Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit Shenyuan Zhang¹, Michael D. Cahalan^{1 1}Department of Physiology and Biophysics, University of California, Irvine (UCI)

• Biology

  
  Whole Cell Recordings from Brain of Adult Drosophila Huaiyu Gu¹, Diane K. O'Dowd^{1 1}Department 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.

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  Induction and Monitoring of Active Delayed Type Hypersensitivity (DTH) in Rats Christine Beeton¹, K. George Chandy^{1 1}Department 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 Flanagan^{1 1}Department 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.

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  Injection of dsRNA into Female A. aegypti Mosquitos Brian M. Luna¹, Jennifer Juhn¹, Anthony A. James^{2 1}Department 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.

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  Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes Olle Terenius¹, Jennifer Juhn¹, Anthony A. James^{2 1}Department 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.

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  Microinjection of A. aegypti Embryos to Obtain Transgenic Mosquitoes Nijole Jasinskiene¹, Jennifer Juhn¹, Anthony A. James^{2 1}Department 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 Chandy^{1 1}Department 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.

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  Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos Beatriz Sicaeros¹, Diane K. O'Dowd^{1 1}Department 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.

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  Dissection of Midgut and Salivary Glands from Ae. aegypti Mosquitoes Judy Coleman¹, Jennifer Juhn¹, Anthony A. James^{2 1}Department 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.

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  Preventing the Spread of Malaria and Dengue Fever Using Genetically Modified Mosquitoes Anthony A. James^{1 1}Department 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. Monuki^{1 1}Department 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.

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  Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae Beatriz Sicaeros¹, Jorge M. Campusano¹, Diane K. O'Dowd^{1 1}Department 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. Hughes^{1 1}Department 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.

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  Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay Souichi Ogata¹, Ken W.Y. Cho^{1 1}Department 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.

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  Plastic Embedding and Sectioning of Xenopus laevis Embryos Souichi Ogata¹, Shimako Kawauchi¹, Anne Calof², Ken W.Y. Cho^{1 1}Department 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. Hughes^{1 1}Department 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.

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  Isolation of Human Umbilical Vein Endothelial Cells (HUVEC) Jaeger Davis¹, Steve P. Crampton¹, Christopher C.W. Hughes^{1 1}Department 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.

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  Harvesting Sperm and Artificial Insemination of Mice Amanda R. Duselis¹, Paul B. Vrana^{1 1}Dept. 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.

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  Retrieval of Mouse Oocytes Amanda R. Duselis¹, Paul B. Vrana^{1 1}Dept. 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.

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  Culture of Mouse Neural Stem Cell Precursors D. Spencer Currle¹, Jia Sheng Hu², Aaron Kolski-Andreaco³, Edwin S. Monuki^{2 1}Department 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 Brachmann^{1 1}Department 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.

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  Growth Factor-Coated Bead Placement on Dorsal Forebrain Explants D. Spencer Currle¹, Aaron Kolski-Andreaco², Edwin S. Monuki^{3 1}Department 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.

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  Mouse Dorsal Forebrain Explant Isolation Spencer Currle¹, Aaron Kolski-Andreaco², Edwin S. Monuki^{3 1}Department 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.

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  Mouse Adrenal Chromaffin Cell Isolation Aaron Kolski-Andreaco¹, Haijiang Cai^2,3, D. Spencer Currle⁴, K. George Chandy¹, Robert H. Chow^{2,3 1}Department 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.