University of California, Los Angeles

50 articles published in JoVE

Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

JoVE 50312 4/30/2013

Department of Physiology, University of California, Los Angeles

In this video, we will demonstrate how to record electrical activity from identified single neurons in a whole brain preparation, which preserves complex neural circuits. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with a fluorescent protein for identification in the intact brain preparation.

Bacterial Detection & Identification Using Electrochemical Sensors

JoVE 4282 4/23/2013

Colin Halford^1,2, Vincent Gau³, Bernard M. Churchill², David A. Haake^2,4,5

¹Research Service, Veterans Affairs Greater Los Angeles Healthcare System, ²Department of Urology, The David Geffen School of Medicine, University of California, Los Angeles, ³GeneFluidics, ⁴Division of Infectious Diseases, Veterans Affairs Greater Los Angeles Healthcare System, ⁵Department of Microbiology, Immunology & Molecular Genetics, University of California, Los Angeles

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Remote Magnetic Navigation for Accurate, Real-time Catheter Positioning and Ablation in Cardiac Electrophysiology Procedures

JoVE 3658 4/21/2013

David Filgueiras-Rama¹, Alejandro Estrada¹, Josh Shachar², Sergio Castrejón¹, David Doiny¹, Marta Ortega¹, Eli Gang³, José L. Merino¹

¹Cardiology, Robotic Cardiac Electrophysiology and Arrhythmia Unit, La Paz University Hospital, ²Magnetecs Corp., ³Cardiology, Geffen School of Medicine at UCLA Los Angeles

This report provides a detailed description of a new remote navigation system based on magnetic driven forces, which has been recently introduced as a new robotic tool for human cardiac electrophysiology procedures.

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

JoVE 50451 4/11/2013

Hongying Zhu¹, Aydogan Ozcan^1,2,3

¹Electrical Engineering Department, University of California, Los Angeles, ²Bioengineering Department, University of California, Los Angeles, ³California NanoSystems Institute (CNSI), University of California, Los Angeles

We review our recent results on the integration of fluorescent microscopy and imaging flow cytometry tools on a cell-phone using compact and cost-effective opto-fluidic attachments. These cell-phone based micro-analysis devices might be useful for cytometric analysis, such as performing various cell counting tasks as well as for high-throughput screening of e.g., water samples in resource limited settings.

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

JoVE 50253 3/11/2013

Bruce Yazejian¹, Rita M. Yazejian¹, Rachel Einarsson², Alan D. Grinnell¹

¹Department of Physiology, David Geffen School of Medicine at UCLA, ²Natural Science Division, Pepperdine University

This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.

Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato

JoVE 50184 1/31/2013

Ana Marie S. Palanca, Alvaro Sagasti

Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles

Optogenetic techniques have made it possible to study the contribution of specific neurons to behavior. We describe a method in larval zebrafish for activating single somatosensory neurons expressing a channelrhodopsin variant (ChEF) with a diode-pumped solid state (DPSS) laser and recording the elicited behaviors with a high-speed video camera.

Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein

JoVE 50169 1/28/2013

Riki Kawaguchi, Ming Zhong, Hui Sun

Department of Physiology, Jules Stein Eye Institute and Howard Hughes Medical Institute, University of California, Los Angeles

Here we describe an optimized technique to produce high-quality vitamin A/RBP complex and two real-time monitoring techniques to study vitamin A transport by STRA6, the RBP receptor.

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method (EFM)

JoVE 4179 12/31/2012

Lawren Sack, Christine Scoffoni

University of California, Los Angeles

We describe a relatively rapid (30 min) and realistic method for simultaneously measurement of leaf hydraulic conductance (K_leaf) and stomatal conductance (g_s) for transpiring excised leaves. The method can be modified to measure the light and dehydration responses of K_leaf and g_s.

Quantitative Analysis of Chromatin Proteomes in Disease

JoVE 4294 12/28/2012

Emma Monte¹, Haodong Chen¹, Maria Kolmakova¹, Michelle Parvatiyar¹, Thomas M. Vondriska^1,2,3, Sarah Franklin^1,4

¹Department of Anesthesiology, David Geffen School of Medicine at UCLA, ²Department of Medicine, David Geffen School of Medicine at UCLA, ³Department of Physiology, David Geffen School of Medicine at UCLA, ⁴Department of Internal Medicine, Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah

Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease.

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

JoVE 4181 12/18/2012

Dimitrios N. Vatakis^1,2,3, Gregory C. Bristol^1,2, Sohn G. Kim^1,2, Bernard Levin^1,2, Wei Liu⁴, Caius G. Radu⁴, Scott G. Kitchen^1,2,3, Jerome A. Zack^1,2,5

¹Department of Medicine, Division of Hematology-Oncology, David Geffen School of Medicine at UCLA, ²UCLA AIDS Institute, ³Eli & Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, ⁴Department of Medical and Molecular Pharmacology, David Geffen School of Medicine at UCLA, ⁵Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine at UCLA

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

Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

JoVE 4221 10/31/2012

David D. Lo*^1,2, Jeong S. Hyun*^1,3, Michael T. Chung¹, Daniel T. Montoro¹, Andrew Zimmermann¹, Monica M. Grova^1,4, Min Lee⁵, Derrick C. Wan¹, Michael T. Longaker¹

¹Department of Surgery, Stanford University, ²Department of Surgery, Duke University, ³Department of Surgery, Saint Joseph Mercy Hospital, ⁴School of Medicine, University of California, San Francisco, ⁵School of Dentistry, University of California, Los Angeles

This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

JoVE 4208 10/01/2012

Hon Wai Koon, Samantha Ho, Michelle Cheng, Ryan Ichikawa, Charalabos Pothoulakis

Center for Inflammatory Bowel Diseases, Division of Digestive Diseases, David Geffen School of Medicine, The University of California Los Angeles, Los Angeles

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

JoVE 3731 9/14/2012

Ahmed E. Hegab, Vi Luan Ha, Yasser S. Attiga, Derek W. Nickerson, Brigitte N. Gomperts

Department of Pediatrics, David Geffen School of Medicine at UCLA

Here we demonstrate our protocol for isolation of basal and submucosal gland duct cells from mouse tracheas. We also demonstrate the method of injecting stem cells into the dorsal mouse fat pad to create an in vivo model of submucosal gland regeneration.

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

JoVE 4391 9/03/2012

Janina Jiang¹, Kathleen A. Kelly^1,2

¹Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, ²California NanoSystems

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution

JoVE 4161 8/16/2012

Serhan O. Isikman¹, Waheb Bishara¹, Aydogan Ozcan^1,2,3

Lensfree optical tomography is a three-dimensional microscopy technique that offers a spatial resolution of <1 μm × <1 μm × <3 μm in x, y and z dimensions, respectively, over a large imaging-volume of 15-100 mm³, which can be particularly useful for integration with lab-on-a-chip platforms.

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus (MRSA) in Rat

JoVE 3863 6/04/2012

Wessam Abdel Hady¹, Arnold S. Bayer^1,2, Yan Q. Xiong^1,2

¹Department of Medicine, Division of Infectious Diseases, Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, ²Geffen School of Medicine at UCLA

Experimental rat endocarditis model due to methicillin-resistant S. aureus.

Aseptic Laboratory Techniques: Volume Transfers with Serological Pipettes and Micropipettors

JoVE 2754 5/31/2012

Erin R. Sanders

Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles

When working in a laboratory, it is imperative to minimize sources of contamination. Aseptic technique refers to procedures that permit transfer of cultures and reagents while avoiding contact with non-sterile surfaces. Serological pipettes and micropipettors are used to measure precise volumes without compromising sterility of solutions used in experiments.

Use of Human Perivascular Stem Cells for Bone Regeneration

JoVE 2952 5/25/2012

Aaron W. James*¹, Janette N. Zara*², Mirko Corselli², Michael Chiang¹, Wei Yuan², Virginia Nguyen¹, Asal Askarinam¹, Raghav Goyal¹, Ronald K. Siu³, Victoria Scott¹, Min Lee³, Kang Ting¹, Bruno Péault^2,4, Chia Soo²

¹Dental and Craniofacial Research Institute and Section of Orthodontics, School of Dentistry, UCLA, ²UCLA and Orthopaedic Hospital, Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, UCLA, ³Department of Bioengineering, UCLA, ⁴Center for Cardiovascular Science, University of Edinburgh

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies

JoVE 3998 5/22/2012

Todd C. Lorenz

Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles

PCR has emerged as a common technique in many molecular biology laboratories. Provided here is a quick guide to several conventional PCR protocols. Because each reaction is a unique experiment, optimal conditions required to generate a product vary. Understanding the variables in a reaction will greatly enhance troubleshooting efficiency, thereby increasing the chance to obtain the desired result.

Aseptic Laboratory Techniques: Plating Methods

JoVE 3064 5/11/2012

Erin R. Sanders

Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles

When working with media and reagents used to culture microorganisms, aseptic technique must be practiced to ensure contamination is minimized. A variety of plating methods are routinely used to isolate, propagate, or enumerate bacteria and phage, all of which incorporate procedures that maintain the sterility of experimental materials.

Agarose Gel Electrophoresis for the Separation of DNA Fragments

JoVE 3923 4/20/2012

Pei Yun Lee, John Costumbrado, Chih-Yuan Hsu, Yong Hoon Kim

Department of Molecular, Cell, and Developmental Biology, University of California Los Angeles

A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

JoVE 3383 1/09/2012

Rachna Ujwal¹, Jeff Abramson²

¹UCLA-DOE Institute for Genomics and Proteomics, University of California Los Angeles, ²Department of Physiology, David Geffen School of Medicine, UCLA

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

Mouse Eye Enucleation for Remote High-throughput Phenotyping

JoVE 3184 11/19/2011

Vinit B. Mahajan^1,2, Jessica M. Skeie^1,2, Amir H. Assefnia^2,3, MaryAnn Mahajan^1,2, Stephen H. Tsang^2,4

¹Department of Ophthalmology and Visual Sciences, University of Iowa, ²Omics Laboratory, University of Iowa, ³School of Dentistry, UCLA, ⁴Bernard and Shirlee Brown Glaucoma Laboratory, Department of Ophthalmology, College of Physicians and Surgeons, Columbia University

The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens.

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

JoVE 3183 10/10/2011

C. Bart Rountree¹, Wei Ding¹, Hein Dang¹, Colleen VanKirk², Gay M. Crooks³

¹Department of Pediatrics and Pharmacology, Pennsylvania State College of Medicine, ²Department of Pharmacology, Pennsylvania State College of Medicine, ³Department of Pediatrics, University of California Los Angeles, School of Medicine

Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.

Lensless Fluorescent Microscopy on a Chip

JoVE 3181 8/17/2011

Ahmet F. Coskun, Ting-Wei Su, Ikbal Sencan, Aydogan Ozcan

Department of Electrical Engineering, University of California, Los Angeles

A lensless on-chip fluorescent microscopy platform is demonstrated that can image fluorescent objects over an ultra-wide field-of-view of e.g., >0.6-8 cm2 with <4μm resolution using a compressive sampling based decoding algorithm. Such a compact and wide-field fluorescent on-chip imaging modality could be valuable for high-throughput cytometry, rare-cell research and microarray-analysis.

Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins

JoVE 2805 7/01/2011

Marija Pinne^1,2, David Haake^3,4

¹Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, ²Research service, 151, Veterans Affairs Greater Los Angeles Healthcare System, ³Departments of Medicine, Urology at David Geffen School of Medicine and Department of Microbiology, Immunology and Molecular Gentics, University of California Los Angeles (UCLA), ⁴Division of Infectious Diseases, 111F, Veterans Affairs Greater Los Angeles Health Care System

An efficient method to assess surface-exposure of leptospiral proteins is described. The method is specifically designed to avoid disruption of the fragile outer membrane of leptospiral cells. This technique requires employment of several negative controls to assess the integrity of the outer membrane and specificity of antibody reaction.

Microwave-assisted One-pot Synthesis of N-succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB)

JoVE 2755 6/28/2011

Shuang Hou*^1,2,3, Duy Linh Phung*^1,2,3, Wei-Yu Lin^1,2,3, Ming-wei Wang⁴, Kan Liu⁵, Clifton Kwang-Fu Shen^1,2,3

¹Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California at Los Angeles, ²Crump Institute for Molecular Imaging, David Geffen School of Medicine, University of California at Los Angeles, ³California NanoSystems Institute, University of California at Los Angeles, ⁴Nuclear Medicine, PET Center, Shanghai Medical Collegea, Fudan University, ⁵Electronics and Information Engineering, College of Electronics and Information Engineering, Wuhan Textile University

A facile, one-pot synthesis of N-succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB) was developed based on a non-aqueous, three-step radiochemical process. Using microwave heating, the entire procedure can be completed in less than 30 min, or 60 min with further purification by preparative HPLC. The decay-corrected radiochemical yields (RCYs) were 35-5% (n > 30).

Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny

JoVE 2559 6/07/2011

Maureen R. Lynch¹, Judith C. Gasson², Helicia Paz¹

¹Department of Medicine, Hematology-Oncology, University of California, Los Angeles, ²Department of Biological Chemistry, University of California, Los Angeles

mStrawberry OP9 cells allow for complete evaluation of all ES-derived progeny from co-culture.

Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method

JoVE 2111 9/06/2010

Han-peng Xu^1,2, Lin Gou^3,4, Hong-Wei Dong³

¹Department of Neurosurgery, Cedars Sinai Medical Center, UCLA, ²Basic Medicine School, Fourth Military Medical University, ³Department of Neurology, David Geffen School of Medicine, UCLA, ⁴Aerospace Medicine School, Fourth Military Medical Univeristy

In this protocol, we described a new method to study the influence of glial cell heterogeneity on axon growth with an in vitro co-culture system. Rat cortical glial cells were cultured to confluence and cocultured with highly purified rat dorsal root ganglia neurons. Different glial cell influence on neurons adhesion and axon growth was compared directly in the same culture. This method provides a new way to directly study the glial cell heterogeneity influence on neuron adhesion and axon growth.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

JoVE 1980 7/27/2010

Colin J. Thalhofer¹, Joel W. Graff², Laurie Love-Homan³, Suzanne M. Hickerson⁴, Noah Craft⁵, Stephen M. Beverley⁴, Mary E. Wilson^6,7

¹Interdisciplinary Immunology Program, University of Iowa, and the VA Medical Center, ²Department of Biochemistry, University of Iowa, and the VA Medical Center, ³Department of Internal Medicine, University of Iowa, ⁴Department of Molecular Microbiology, Washington University School of Medicine, ⁵Division of Dermatology, Harbor-UCLA Medical Center, Hanley-Hardison Research Center, ⁶Interdisciplinary Immunology Program, Iowa City VA Medical Center, ⁷Departments of Internal Medicine, Microbiology and Epidemiology, University of Iowa

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

Low-stress Route Learning Using the Lashley III Maze in Mice

JoVE 1786 5/22/2010

Amanda Bressler¹, David Blizard², Anne Andrews^3,4,5,6

¹Department of Chemistry, Pennsylvania State University, ²Center for Developmental and Health Genetics, Pennsylvania State University, ³Department of Veterinary and Biomedical Sciences, Pennsylvania State University, ⁴Huck Institute of the Life Sciences, Pennsylvania State University, ⁵California NanoSystems Institute, University of California, Los Angeles, ⁶Semel Institute of Neuroscience and Human Behavior, University of California, Los Angeles

The Lashley III maze is a route-learning task that does not rely on aversive stimuli or visual cues. It is thus a highly attractive option for evaluating learning and memory, especially in aging mice or otherwise where stress is a consideration.

Selection of Aptamers for Amyloid β-Protein, the Causative Agent of Alzheimer's Disease

JoVE 1955 5/13/2010

Farid Rahimi¹, Gal Bitan^1,2,3

¹Department of Neurology, David Geffen School of Medicine, ²Molecular Biology Institute, University of California, Los Angeles, ³Brain Research Institute, University of California, Los Angeles

Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid β-protein, the causative agent of Alzheimer's disease.

Lensless On-chip Imaging of Cells Provides a New Tool for High-throughput Cell-Biology and Medical Diagnostics

JoVE 1650 12/14/2009

Onur Mudanyali¹, Anthony Erlinger¹, Sungkyu Seo¹, Ting-Wei Su¹, Derek Tseng¹, Aydogan Ozcan^1,2

¹Electrical Engineering Department, University of California, Los Angeles, ²California NanoSystems Institute, University of California, Los Angeles

Lensfree on-chip imaging and characterization of cells is illustrated. This on-chip cell imaging approach provides a compact and cost-effective tool for medical diagnostics and high-throughput cell biology applications, making it especially suitable for resource poor settings.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

JoVE 1520 10/19/2009

Marino DiFranco, Marbella Quinonez, Joana Capote, Julio Vergara

Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

JoVE 1355 6/08/2009

Yali Zhao¹, Dan O. Wang¹, Kelsey C. Martin^1,2,3

¹Dept. of Psychiatry and Biobehavioral Sciences, University of California, Los Angeles, ²Dept. of Biological Chemistry, University of California, Los Angeles, ³Semel Institute for Neuroscience and Human Behavior, University of California, Los Angeles

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

JoVE 1178 5/18/2009

Harpreet Singh¹, Sarah Warburton², Thomas M. Vondriska³, Baljit S. Khakh¹

¹Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, ²Department of Anesthesiology, David Geffen School of Medicine, University of California, Los Angeles, ³Department of Anesthesiology, Medicine and Physiology, David Geffen School of Medicine, University of California, Los Angeles

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

JoVE 1142 4/26/2009

Eiji Shigetomi, Baljit S. Khakh

Departments of Physiology and Neurobiology, David Geffen School of Medicine, University of California, Los Angeles

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

JoVE 1129 2/16/2009

Georgeann S. O'Brien¹, Sandra Rieger¹, Seanna M. Martin¹, Ann M. Cavanaugh¹, Carlos Portera-Cailliau², Alvaro Sagasti¹

¹Department of Molecular Cell and Developmental Biology, University of California, Los Angeles, ²Departments of Neurology and Neurobiology, University of California, Los Angeles

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

Visually Mediated Odor Tracking During Flight in Drosophila

JoVE 1110 1/26/2009

Mark A. Frye, Brian J. Duistermars

Department of Physiological Science, University of California, Los Angeles

Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.

Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides

JoVE 1071 1/12/2009

Farid Rahimi¹, Panchanan Maiti¹, Gal Bitan^2,3

¹Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, ²Brain Research Institute, Molecular Biology Institute, University of California, Los Angeles, ³Department of Neurology, University of California, Los Angeles

Photo-induced cross-linking of unmodified proteins (PICUP) allows characterization of oligomer size distribution in metastable protein mixtures. We demonstrate application of PICUP to three representative amyloidogenic peptides the 40- and 42-residue forms of amyloid β-protein, and calcitonin, and a control peptide growth-hormone releasing factor.

A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila

JoVE 1063 11/21/2008

Brian J. Duistermars, Mark A. Frye

Department of Physiological Science, University of California, Los Angeles

Here we describe how to tether a fly in an olfactory magnetic-tether (OMT) apparatus. We describe how to align the rare-earth magnets and odor ports, and how to set mass flow rates for both the stimulus delivery and vacuum suction to achieve optimal odor tracking.

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

JoVE 742 6/24/2008

Christos Gekas, Katrin E. Rhodes, Hanna K. A. Mikkola

Jonsson Comprehensive Cancer Center, University of California, Los Angeles

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

JoVE 724 6/17/2008

Ivan Khvorostov, Jin Zhang, Michael Teitell

David Geffen School of Medicine, University of California, Los Angeles

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel

JoVE 832 6/10/2008

Jin Zhang, Ivan Khvorostov, Michael Teitell

David Geffen School of Medicine, University of California, Los Angeles

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 3 of 3.

From MEFs to Matrigel 2: Splitting hESCs from MEFs onto Matrigel

JoVE 831 6/09/2008

Ivan Khvorostov, Jin Zhang, Michael Teitell

David Geffen School of Medicine, University of California, Los Angeles

From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs

JoVE 722 6/04/2008

Jin Zhang, Ivan Khvorostov, Michael Teitell

David Geffen School of Medicine, University of California, Los Angeles

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells. Part 1 of 3.

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

JoVE 681 3/13/2008

Peyman Golshani, Carlos Portera-Cailliau

Department of Neurology, University of California, Los Angeles

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window

JoVE 678 2/25/2008

Ricardo Mostany, Carlos Portera-Cailliau

Department of Neurology, University of California, Los Angeles

Cortical blood flow dynamics can be studied in vivo by imaging fluorescent dextran dyes injected into the tail vein of rodents with 2-photon microscopy. This video shows how to image blood flow dynamics in neocortex of mice through a glass-covered cranial window preparation.

A Craniotomy Surgery Procedure for Chronic Brain Imaging

JoVE 680 2/15/2008

Ricardo Mostany, Carlos Portera-Cailliau

Department of Neurology, University of California, Los Angeles

This video and protocol demonstrate how to implant a glass-covered cranial window in rodents. These preparations can be used for chronic in vivo two-photon imaging of the neocortex over time scales of months. It may also be used for other types of imaging, including optical intrinsic signal imaging.

Predicting the Effectiveness of Population Replacement Strategy Using Mathematical Modeling

JoVE 227 7/04/2007

John Marshall, Koji Morikawa, Nicholas Manoukis, Charles Taylor

Department of Ecology and Evolutionary Biology, University of California, Los Angeles

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy. Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies. The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.