Harvard University View Institution's Website 127 articles published in JoVE Bioengineering Modeling Healthy and Dysbiotic Vaginal Microenvironments in a Human Vagina-on-a-Chip Aakanksha Gulati1, Alicia Jorgenson1, Abidemi Junaid1, Donald E. Ingber1,2,3 1Wyss Institute for Biologically Inspired Engineering, Harvard University, 2 This article describes a protocol for creating a microfluidic vagina-on-a-chip (Vagina Chip) culture device that enables the study of human host interactions with a living vaginal microbiome under microaerophilic conditions. This chip can be used as a tool to investigate vaginal diseases as well as to develop and test potential therapeutic countermeasures. Environment Coral Reef Arks: An In Situ Mesocosm and Toolkit for Assembling Reef Communities Jason L. Baer1, Jessica Carilli2, Bart Chadwick3, Mark Hatay1, Anneke van der Geer1, Yun Scholten4, William Barnes4, Jenna Aquino1, Ashton Ballard1, Mark Little1, Jared Brzenski5, Xiaofeng Liu6, Gunther Rosen2, Pei-Fang Wang2, Jose Castillo5, Andreas F. Haas4, Aaron C. Hartmann7, Forest Rohwer1 1Department of Biology, San Diego State University, 2Energy and Environmental Sciences Branch, Naval Information Warfare Center (NIWC) Pacific, 3Coastal Monitoring Associates, 4Department of Marine Microbiology and Biogeochemistry, NIOZ Royal Netherlands Institute for Sea Research, 5Computational Science Research Center, San Diego State University, 6Department of Aerospace Engineering, San Diego State University, 7Department of Organismic and Evolutionary Biology, Harvard University Moored midwater geodesic structures called Coral Arks provide a modular, scalable, and vertically adjustable research platform that can be used to build, monitor, and perturb coral reef communities in previously inoperative areas, including offshore. Engineering Triplet Fusion Upconversion Nanocapsule Synthesis Tracy H. Schloemer1,2, Samuel N. Sanders1, Qi Zhou2, Pournima Narayanan3, Manchen Hu2, Mahesh K. Gangishetty1, Daniel Anderson1, Michael Seitz1,2, Arynn O. Gallegos2, R. Christopher Stokes1, Daniel N. Congreve1,2 1Rowland Institute, Harvard University, 2Department of Electrical Engineering, Stanford University, 3Department of Chemistry, Stanford University This protocol details the synthesis of upconversion nanocapsules for subsequent use in photopolymerizable resins for triplet fusion upconversion-facilitated volumetric 3D printing. Bioengineering Rapid Encapsulation of Reconstituted Cytoskeleton Inside Giant Unilamellar Vesicles Yashar Bashirzadeh*1, Nadab Wubshet*1, Thomas Litschel2, Petra Schwille3, Allen P. Liu1,4,5,6 1Department of Mechanical Engineering, University of Michigan, Ann Arbor, 2John A. Paulson School of Engineering and Applied Sciences, Harvard University, 3Department of Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, 4Department of Biomedical Engineering, University of Michigan, Ann Arbor, 5Department of Biophysics, University of Michigan, Ann Arbor, 6Cellular and Molecular Biology Program, University of Michigan, Ann Arbor This article introduces a simple method for expeditious production of giant unilamellar vesicles with encapsulated cytoskeletal proteins. The method proves to be useful for bottom-up reconstitution of cytoskeletal structures in confinement and cytoskeleton-membrane interactions. Medicine 3D Imaging of PDL Collagen Fibers during Orthodontic Tooth Movement in Mandibular Murine Model Han Xu1, Andy Lee1, Lu Sun1, Gili R. S. Naveh1 1Department of Oral Medicine, Infection and Immunity, School of Dental Medicine, Harvard University We present a protocol for generating orthodontic tooth movement in mice and methods for 3D visualization of the collagen fibers and blood vessels of periodontal ligament without sectioning. Biology Inhibition of Wound Epidermis Formation via Full Skin Flap Surgery During Axolotl Limb Regeneration Stephanie Tsai1,2 1Department of Stem Cell and Regenerative Biology, Harvard University, 2Department of Molecular and Cellular Biology, Harvard University This article describes how to perform a surgical method to inhibit wound epidermis formation during axolotl limb regeneration by immediately suturing full thickness skin over the amputation plane. This method allows researchers to investigate the functional roles of the wound epidermis during the early stages of limb regeneration. Developmental Biology Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis Eileen F. Ablondi1, Sudip Paudel2, Morgan Sehdev3, John P. Marken4, Andrew D. Halleran4, Atiqur Rahman5, Peter Kemper5, Margaret S. Saha2 1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Harvard University, 2Department of Biology, College of William and Mary, 3Harvard Medical School, Harvard University, 4Department of Bioengineering, California Institute of Technology, 5Department of Computer Science, College of William and Mary We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level. Behavior A Methodology for Capturing Joint Visual Attention Using Mobile Eye-Trackers Bertrand Schneider1 1Learning, Innovation, and Technology Lab, Graduate School of Education, Harvard University Using multimodal sensors is a promising way to understand the role of social interactions in educational settings. This paper describes a methodology for capturing joint visual attention from colocated dyads using mobile eye-trackers. Developmental Biology Injecting Gryllus bimaculatus Eggs Samantha K. Barry1, Taro Nakamura2, Yuji Matsuoka3, Christoph Straub4, Hadley W. Horch4, Cassandra G. Extavour5 1Colby College, 2Division of Evolutionary Developmental Biology, National Institute for Basic Biology, 3Department of Biological Sciences, National University of Singapore, 4Department Biology and Department of Neuroscience, Bowdoin College, 5Department of Organismic and Evolutionary Biology and Department of Molecular and Cellular Biology, Harvard University Here we present a protocol to inject cricket eggs, a technique which serves as a foundational method in many experiments in the cricket, including, but not limited to, RNA interference and genomic manipulation. Genetics Designing Automated, High-throughput, Continuous Cell Growth Experiments Using eVOLVER Zachary J. Heins1,2, Christopher P. Mancuso1,2, Szilvia Kiriakov1,3, Brandon G. Wong1,2, Caleb J. Bashor4, Ahmad S. Khalil1,2,5 1Biological Design Center, Boston University, 2Department of Biomedical Engineering, Boston University, 3Program in Molecular Biology, Cell Biology and Biochemistry, Boston University, 4Department of Bioengineering, Rice University, 5Wyss Institute for Biologically Inspired Engineering, Harvard University The eVOLVER framework enables high-throughput continuous microbial culture with high resolution and dynamic control over experimental parameters. This protocol demonstrates how to apply the system to conduct a complex fitness experiment, guiding users on programming automated control over many individual cultures, measuring, collecting, and interacting with experimental data in real-time. Environment Production and Measurement of Organic Particulate Matter in a Flow Tube Reactor Yue Zhang1,2, Pengfei Liu1, Zhaoheng Gong1, Franz M. Geiger3, Scot T. Martin1,4 1School of Engineering and Applied Sciences, Harvard University, 2Department of Environmental Science and Engineering, Gillings School of Global Public Health, University of North Carolina, Chapel Hill, 3Department of Chemistry, Northwestern University, 4Department of Earth and Planetary Sciences, Harvard University This paper describes the operation procedure for the flow tube reactor and related data collection. It shows the protocols for setting the experiments, recording data and generating the number-diameter distribution as well as the particle mass information, which gives useful information about chemical and physical properties of the organic aerosols. Environment Production and Measurement of Organic Particulate Matter in the Harvard Environmental Chamber Yue Zhang1,2, Zhaoheng Gong1, Suzane de Sa1, Adam P. Bateman1, Yingjun Liu1, Yongjie Li1, Franz M. Geiger3, Scot T. Martin1,4 1School of Engineering and Applied Sciences, Harvard University, 2Department of Environmental Science and Engineering, Gillings School of Global Public Health, University of North Carolina, 3Department of Chemistry, Northwestern University, 4Department of Earth and Planetary Sciences, Harvard University This paper describes operation procedures for the Harvard Environmental Chamber (HEC) and related instrumentation for measuring gaseous and particle species. The environmental chamber is used to produce and study secondary organic species produced from the organic precursors, especially related to atmospheric organic particulate matter. Neuroscience A Micro-CT-based Method for Characterizing Lesions and Locating Electrodes in Small Animal Brains Javier Masis1,2, David Mankus2, Steffen B.E. Wolff2,3, Grigori Guitchounts1,2, Maximilian Joesch4, David D. Cox1,2 1Department of Molecular and Cellular Biology, Harvard University, 2Center for Brain Science, Harvard University, 3Department of Organismic and Evolutionary Biology, Harvard University, 4Institute of Science and Technology Austria This article describes a straightforward method to prepare small animal brains for micro-CT imaging, in which lesions can be quantified and electrodes located with high precision in the context of the whole brain. Bioengineering Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips Richard Novak*1, Meredyth Didier*1,2, Elizabeth Calamari1, Carlos F Ng1, Youngjae Choe1, Susan L Clauson1, Bret A Nestor1, Jefferson Puerta1, Rachel Fleming1, Sasan J Firoozinezhad1, Donald E Ingber1,3,4 1Wyss Institute for Biologically Inspired Engineering, Harvard University, 2Apple, Inc, 3Harvard John A. Paulson School of Engineering and Applied Sciences, Harvard University, 4 Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro. Genetics CRISPR Guide RNA Cloning for Mammalian Systems Sathiji Nageshwaran*1,2, Alejandro Chavez*1,2,3, Nan Cher Yeo1,2, Xiaoge Guo1,2, Alissa Lance-Byrne1, Angela Tung1, James J. Collins1,4,5,6,7, George M. Church1,2 1Wyss Institute for Biologically Inspired Engineering, Harvard University, 2Department of Genetics, Harvard Medical School, 3Department of Pathology, Massachusetts General Hospital, 4Institute for Medical Engineering & Science, Massachusetts Institute of Technology, 5Synthetic Biology Center, Massachusetts Institute of Technology, 6Department of Biological Engineering, Massachusetts Institute of Technology, 7Broad Institute Here, a simple, efficient, and cost-effective method of sgRNA cloning is outlined. Bioengineering Syringe-injectable Mesh Electronics for Stable Chronic Rodent Electrophysiology Thomas G. Schuhmann Jr.1, Tao Zhou2, Guosong Hong2, Jung Min Lee2,3, Tian-Ming Fu2, Hong-Gyu Park3, Charles M. Lieber1,2 1John A. Paulson School of Engineering and Applied Sciences, Harvard University, 2Department of Chemistry and Chemical Biology, Harvard University, 3Department of Physics, Korea University Mesh electronics probes seamlessly integrate and provide stable, long-term, single-neuron level recording within the brain. This protocol uses mesh electronics for in vivo experiments, involving the fabrication of mesh electronics, loading into needles, stereotaxic injection, input/output interfacing, recording experiments, and histology of tissue containing mesh probes. Bioengineering A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans Kyung Suk Lee1,2, Erel Levine2,3 1Department of Physics Education, Kongju National University, 2Department of Physics, Harvard University, 3FAS Center for Systems Biology, Harvard University In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. Chemistry Synthesis and Performance Characterizations of Transition Metal Single Atom Catalyst for Electrochemical CO2 Reduction Kun Jiang1, Guangxu Chen2, Haotian Wang1 1Rowland Institute, Harvard University, 2Materials Science and Engineering, Stanford University Here, we present a protocol for the synthesis and electrochemical testing of transition metal single atoms coordinated in graphene vacancies as active centers for selective carbon dioxide reduction to carbon monoxide in aqueous solutions. Biology Single-cell Microfluidic Analysis of Bacillus subtilis Matthew T. Cabeen1, Richard Losick2 1Department of Microbiology and Molecular Genetics, Oklahoma State University, 2Department of Molecular and Cellular Biology, Harvard University We present a method for the microfluidic analysis of individual bacterial cell lineages using Bacillus subtilis as an example. The method overcomes shortcomings of traditional analytical methods in microbiology by allowing observation of hundreds of cell generations under tightly controllable and uniform growth conditions. Neuroscience Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids Max R Salick*1, Michael F Wells*2,3, Kevin Eggan2,3, Ajamete Kaykas1 1Department of Neuroscience, Novartis Institutes for BioMedical Research, 2Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, 3Department of Stem Cell and Regenerative Biology and Harvard Stem Cell Institute, Harvard University This protocol describes a technique used to model Zika virus infection of the developing human brain. Using wildtype or engineered stem cell lines, researchers may use this technique to uncover the various mechanisms or treatments that may affect early brain infection and resulting microcephaly in Zika virus-infected embryos. Neuroscience External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures Shani Stern1, Assaf Rotem2, Yuri Burnishev3, Eyal Weinreb3, Elisha Moses3 1Laboratory of Genetics, The Salk Institute for Biological Studies, 2Department of Physics and SEAS, Harvard University, 3Department of Physics of Complex Systems, Weizmann Institute of Science Neuronal cultures are a good model for studying emerging brain stimulation techniques via their effect on single neurons or a population of neurons. Presented here are different methods for stimulation of patterned neuronal cultures by an electric field produced directly by bath electrodes or induced by a time-varying magnetic field. Bioengineering Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device Hyun Jung Kim1, Jaewon Lee1, Jin-Ha Choi1, Anthony Bahinski2, Donald E. Ingber2,3,4 1Department of Biomedical Engineering, The University of Texas at Austin, 2Wyss Institute for Biologically Inspired Engineering at Harvard University, 3 We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system. Medicine Unbiased Deep Sequencing of RNA Viruses from Clinical Samples Christian B. Matranga1, Adrianne Gladden-Young1, James Qu1, Sarah Winnicki1, Dolo Nosamiefan1, Joshua Z. Levin1, Pardis C. Sabeti1,2 1Broad Institute of MIT and Harvard, 2Harvard University This protocol describes a rapid and broadly applicable method for unbiased RNA-sequencing of viral samples from human clinical isolates. Immunology and Infection Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria Rachel Daniels1,2, Elizabeth J. Hamilton2, Katelyn Durfee2, Daouda Ndiaye3, Dyann F. Wirth2,5, Daniel L. Hartl1, Sarah K. Volkman2,4 1Department of Organismic and Evolutionary Biology, Harvard University, 2Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, 3Faculty of Medicine and Pharmacy, Cheikh Anta Diop University, 4School of Nursing and Health Sciences, Simmons College, 5Institute of Infectious Diseases, Broad Institute While high resolution melting analysis offers the ability to differentiate between single nucleotide polymorphisms in a heterogeneous population, mutant allele amplification bias can increase its ability to detect alleles present at relatively low percentages within a sample. This protocol describes improvements that improve the sensitivity of high resolution melting analysis. Engineering Making Record-efficiency SnS Solar Cells by Thermal Evaporation and Atomic Layer Deposition Rafael Jaramillo2,4, Vera Steinmann1,2, Chuanxi Yang3, Katy Hartman2,4, Rupak Chakraborty1,2, Jeremy R. Poindexter2,4, Mariela Lizet Castillo2, Roy Gordon5, Tonio Buonassisi1,2 1Department of Mechanical Engineering, Massachusetts Institute of Technology, 2Laboratory for Manufacturing and Productivity, Massachusetts Institute of Technology, 3School of Engineering and Applied Sciences, Harvard University, 4Department of Materials Science and Engineering, Massachusetts Institute of Technology, 5Department of Chemistry & Chemical Biology, Harvard University Tin sulfide (SnS) is a candidate material for Earth-abundant, non-toxic solar cells. Here, we demonstrate the fabrication procedure of the SnS solar cells employing atomic layer deposition, which yields 4.36% certified power conversion efficiency, and thermal evaporation which yields 3.88%. Chemistry Self-assembly of Complex Two-dimensional Shapes from Single-stranded DNA Tiles Bryan Wei1, Michelle K. Vhudzijena2, Joanna Robaszewski2, Peng Yin2,3 1Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, 2Wyss Institute for Biologically Inspired Engineering, Harvard University, 3Department of Systems Biology, Harvard Medical School DNA tiling is an effective approach to make programmable nanostructures. We describe the protocols to construct complex two-dimensional shapes by the self-assembly of single-stranded DNA tiles. Developmental Biology Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP) Katherine W. Rogers1, Alexander Bläßle2, Alexander F. Schier1, Patrick Müller2 1Department of Molecular and Cellular Biology, Harvard University, 2Systems Biology of Development Group, Friedrich Miescher Laboratory of the Max Planck Society Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo. Bioengineering Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery Dwayne A. L. Vickers1, Soojung Claire Hur1 1The Rowland Institute, Harvard University A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system’s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability. Medicine Ultrasound-guided Transthoracic Intramyocardial Injection in Mice Terence W. Prendiville1, Qing Ma1, Zhiqiang Lin1, Pingzhu Zhou1, Aibin He1, William T. Pu1,2 1Department of Cardiology, Boston Children's Hospital, 2Harvard Stem Cell Institute, Harvard University Echocardiography-guided percutaneous intramyocardial injection represents an efficient, reliable, and targetable modality for the delivery of gene transfer agents or cells into the murine heart. Following the steps outlined in this protocol, the operator can quickly become competent in this versatile, minimally invasive technique. Environment Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun Matthew D. Mattozzi1,2, Mathias J. Voges1,2,3, Pamela A. Silver1,2, Jeffrey C. Way1,2 1Synthetic Biology Platform, Wyss Institute for Biologically Inspired Engineering, Harvard University, 2Department of Systems Biology, Harvard Medical School, 3Department of Biotechnology, Delft University of Technology This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun. Biology Facial Transplants in Xenopus laevis Embryos Laura A. Jacox*1,3, Amanda J. Dickinson*4, Hazel Sive2,3 1Biological Sciences in Dental Medicine, Harvard University, 2Biology Department, Massachusetts Institute of Technology, 3Whitehead Institute, Massachusetts Institute of Technology, 4Biology Department, Virginia Commonwealth University A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions. Biology Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis Anastasia R. Nast1, Cassandra G. Extavour1 1Department of Organismic and Evolutionary Biology, Harvard University The amphipod Parhyale hawaiensis is a promising model organism for studies of crustacean embryology and comparative arthropod development and evolution. This protocol describes a method for manual removal of single blastomeres from early cleavage stage embryos of Parhyale. Biology Digital Inline Holographic Microscopy (DIHM) of Weakly-scattering Subjects Camila B. Giuliano1,2, Rongjing Zhang1, Laurence G. Wilson1 1The Rowland Institute, Harvard University, 2Faculdade de Ciências e Letras de Assis, Universidade Estadual Paulista The three-dimensional locations of weakly-scattering objects can be uniquely identified using digital inline holographic microscopy (DIHM), which involves a minor modification to a standard microscope. Our software uses a simple imaging heuristic coupled with Rayleigh-Sommerfeld back-propagation to yield the three-dimensional position and geometry of a microscopic phase object. Biology Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays Laura L. Smith1, Alan H. Beggs1, Vandana A. Gupta1 1Division of Genetics and Genomics, Manton Center for Orphan Disease Research, Boston Children's Hospital, Harvard Medical School The zebrafish is now an established and powerful tool for modeling muscular dystrophies, congenital myopathies, and related neuromuscular diseases. Birefringence and touch-evoked escape behavior are two common noninvasive assays used to determine the degree of muscular disorganization and locomotive impairment of zebrafish embryos during early development. Bioengineering Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System Oren Levy1,2,3,4,5, Priya Anandakumaran1,2,3,4,5, Jessica Ngai1,2,3,4,5, Rohit Karnik6, Jeffrey M. Karp1,2,3,4,5 1 This study used a multi-well plate microfluidic system, significantly increasing throughput of cell rolling studies under physiologically relevant shear flow. Given the importance of cell rolling in the multi-step cell homing cascade and the importance of cell homing following systemic delivery of exogenous populations of cells in patients, this system offers potential as a screening platform to improve cell-based therapy. Biology Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland Silva Krause1, Amy Brock2, Donald E. Ingber1,2,3 1 A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules. Immunology and Infection In vivo Imaging Method to Distinguish Acute and Chronic Inflammation Jen-Chieh Tseng1, Andrew L. Kung2 1Lurie Family Imaging Center, Dana-Farber Cancer Institute, Harvard Medical School, 2Division of Pediatric Hematology/Oncology/Stem Cell Transplantation, Columbia University Medical Center We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages. Biology Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs Judith A. Sharp1,2, Mike D. Blower1,2 1Department of Molecular Biology, Massachusetts General Hospital, 2Department of Genetics, Harvard Medical School We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs. Behavior Simultaneous EEG Monitoring During Transcranial Direct Current Stimulation Pedro Schestatsky1,2,3, Leon Morales-Quezada3,4, Felipe Fregni3 1Programa de Pós-Graduação em Ciências Médica, Universidade Federal do Rio Grande do Sul, 2Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES), 3Laboratory of Neuromodulation, Department of Physical Medicine & Rehabilitation, Spaulding Rehabilitation Hospital and Massachusetts General Hospital, Harvard Medical School, 4De Montfort University Transcranial direct current stimulation (tDCS) is a non-invasive brain stimulation technique that has shown initial therapeutic effects in several neurological conditions. The main mechanism underlying these therapeutic effects is the modulation of cortical excitability. Therefore, online monitoring of cortical excitability would help guide stimulation parameters and optimize its therapeutic effects. In the present article we review the use of a novel device that combines simultaneous tDCS and EEG monitoring in real time. Biology Assessing Murine Resistance Artery Function Using Pressure Myography Mohd Shahid1, Emmanuel S. Buys1 1Anesthesia Center for Critical Care Research, Department of Anesthesia, Critical Care, and Pain Medicine, Massachusetts General Hospital, Harvard Medical School In pressure myography, an intact small segment of a vessel is mounted onto two small cannulas and pressurized to a suitable luminal pressure. Here, we describe the method to measure vasorelaxation response of the mouse 3rd order mesenteric arteries in c57 and sGCα1-/- mice using pressure myography. Biology Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans Elizabeth C. Pino1, Christopher M. Webster1, Christopher E. Carr2, Alexander A. Soukas1 1Center for Human Genetic Research and Department of Medicine, Massachusetts General Hospital and Harvard Medical School, 2Department of Earth, Atmospheric, and Planetary Sciences, Massachusetts Institute of Technology We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry. Medicine Development of an Audio-based Virtual Gaming Environment to Assist with Navigation Skills in the Blind Erin C. Connors1, Lindsay A. Yazzolino1, Jaime Sánchez2, Lotfi B. Merabet1 1Laboratory for Visual Neuroplasticity, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, 2Department of Computer Science and Center for Advanced Research in Education (CARE), University of Chile Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Bioengineering Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing Ayhan Atmanli1, Ibrahim J. Domian1,2 1Cardiovascular Research Center, Massachusetts General Hospital and Harvard Medical School, 2Harvard Stem Cell Institute The generation of aligned myocardial tissue is a key requirement for adapting the recent advances in stem cell biology to clinically useful purposes. Herein we describe a microcontact printing approach for the precise control of cell shape and function. Using highly purified populations of embryonic stem cell derived cardiac progenitors, we then generate anisotropic functional myocardial tissue. Medicine Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants Pei-Yi Lin1, Nadege Roche-Labarbe1,2, Mathieu Dehaes3, Stefan Carp1, Angela Fenoglio3, Beniamino Barbieri4, Katherine Hagan1, P. Ellen Grant3, Maria Angela Franceschini1 1Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital, Harvard Medical School, 2Lab. PALM, Université de Caen Basse-Normandie, 3Fetal-Neonatal Neuroimaging and Developmental Science Center, Boston Children's Hospital, Harvard Medical School, 4ISS, INC. We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain. Neuroscience Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants Mauro W. Zappaterra1, Anthony S. LaMantia2, Christopher A. Walsh3,4, Maria K. Lehtinen5 1Department of Physical Medicine and Rehabilitation, VA Greater Los Angeles Healthcare System, 2Department of Pharmacology and Physiology, Institute for Neuroscience, The George Washington University School of Medicine and Health Sciences, 3Division of Genetics, Department of Medicine, Boston Children's Hospital, 4Howard Hughes Medical Institute, Boston Children's Hospital, 5Department of Pathology, Boston Children's Hospital, Harvard Medical School The ventricular cerebrospinal fluid (CSF) bathes the neuroepithelial and cerebral cortical progenitor cells during early brain development in the embryo. Here we describe the method developed to isolate ventricular CSF from rodent embryos of different ages in order to investigate its biological function. In addition, we demonstrate our cerebral cortical explant dissection and culture technique that allows for explant growth with minimal volumes of culture medium or CSF. Bioengineering Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation Lida P. Hariri1,2,3, Matthew B. Applegate4, Mari Mino-Kenudson1,2, Eugene J. Mark1,2, Brett E. Bouma2,3, Guillermo J. Tearney1,2,3, Melissa J. Suter2,3,5 1Department of Pathology, Harvard Medical School, 2Massachusetts General Hospital, 3Wellman Center for Photomedicine, Harvard Medical School, 4Pulmonary and Critical Care Unit, Massachusetts General Hospital, 5Pulmonary and Critical Care Unit, Harvard Medical School A method to image ex vivo pulmonary resection specimens with optical frequency domain imaging (OFDI) and obtain precise correlation to histology is described, which is essential to developing specific OFDI interpretation criteria for pulmonary pathology. This method is applicable to other tissue types and imaging techniques to obtain precise imaging to histology correlation for accurate image interpretation and assessment. Imaging criteria established with this technique would then be applicable to image assessment in future in vivo studies. Engineering A Method to Fabricate Disconnected Silver Nanostructures in 3D Kevin Vora1, SeungYeon Kang1, Eric Mazur1,2 1School of Engineering and Applied Sciences, Harvard University, 2Department of Physics, Harvard University Femtosecond-laser direct-writing is frequently used to create three-dimensional (3D) patterns in polymers and glasses. However, patterning metals in 3D remains a challenge. We describe a method for fabricating silver nanostructures embedded inside a polymer matrix using a femtosecond laser centered at 800 nm. Neuroscience Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals Timothy M. Otchy1,2, Bence P. Ӧlveczky1,3 1Center for Brain Science, Harvard University, 2Program in Neuroscience, Harvard University, 3Department of Organismic and Evolutionary Biology, Harvard University The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals. Immunology and Infection Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells Francois P. Legoux1, James J. Moon1 1Center for Immunology and Inflammatory Diseases, and Pulmonary and Critical Care Unit, Massachusetts General Hospital and Harvard Medical School This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems. Neuroscience Viral Tracing of Genetically Defined Neural Circuitry Kevin Beier1, Constance Cepko2 1Department of Genetics, Harvard Medical School, 2Howard Hughes Medical Institute, Harvard Medical School A method of tracing synaptically connected neurons is described. We use TVA specificity of an upstream cell to probe whether a cell population of interest receives synaptic input from genetically defined cell types. Neuroscience Neural Explant Cultures from Xenopus laevis Laura Anne Lowery1, Anna E.R. Faris1, Alina Stout1, David Van Vactor1 1Department of Cell Biology, Harvard Medical School Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics. Bioengineering Evaluation of Biomaterials for Bladder Augmentation using Cystometric Analyses in Various Rodent Models Duong D. Tu*1, Abhishek Seth*1, Eun Seok Gil2, David L. Kaplan2, Joshua R. Mauney1, Carlos R. Estrada Jr.1 1Children's Hospital Boston, Harvard Medical School, 2Tufts University Surgical stages of bladder augmentation are described using 3-D scaffolds in murine and rat models. To test the efficacy of biomaterial configurations for use in bladder augmentation, techniques for both awake and anesthetized cystometry are presented. Immunology and Infection Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation Tatiana Veremeyko1, Sarah-Christine Starossom1, Howard L. Weiner1, Eugene D. Ponomarev1 1Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia. Biology Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries Eric G. Folco1, Haixin Lei1, Jeanne L. Hsu1, Robin Reed1 1Department of Cell Biology, Harvard Medical School A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells. Biology Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans Stephen A. Banse1, Craig P. Hunter1 1Department of Molecular and Cellular Biology, Harvard University The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example. Medicine In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis Rachel A. Davidowitz1, Marcin P. Iwanicki1, Joan S. Brugge1 1Department of Cell Biology, Harvard Medical School The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis. Biology Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction Xuejun H. Parsons1,2, Yang D. Teng3,4, James F. Parsons1,2, Evan Y. Snyder1,2,5, David B. Smotrich1,2,6, Dennis A. Moore1,2 1San Diego Regenerative Medicine Institute, 2Xcelthera, 3Department of Neurosurgery, Harvard Medical School, 4Division of SCI Research, VA Boston Healthcare System, 5Program in Stem Cell & Regenerative Biology, Sanford-Burnham Medical Research Institute, 6La Jolla IVF We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair. Neuroscience Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction Xuejun H. Parsons1,2, Yang D. Teng3,4, James F. Parsons1,2, Evan Y. Snyder1,2,5, David B. Smotrich1,2,6, Dennis A. Moore1,2 1San Diego Regenerative Medicine Institute, 2Xcelthera, 3Department of Neurosurgery, Harvard Medical School, 4Division of SCI Research, VA Boston Healthcare System, 5Program in Stem Cell & Regenerative Biology, Sanford-Burnham Medical Research Institute, 6La Jolla IVF We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair. Bioengineering Video-rate Scanning Confocal Microscopy and Microendoscopy Alexander J. Nichols1,2,3, Conor L. Evans1,3 1Program in Biophysics, Harvard University, 2Division of Health Sciences and Technology, Harvard-MIT, 3Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School The complete construction of a custom, real-time confocal scanning imaging system is described. This system, which can be readily used for video-rate microscopy and microendoscopy, allows for an array of imaging geometries and applications not accessible using standard commercial confocal systems, at a fraction of the cost. Immunology and Infection RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract Eduardo Lopez-Medina1, Megan M. Neubauer1, Gerald B. Pier2, Andrew Y. Koh3 1Department of Pediatrics, University of Texas Southwestern Medical Center, 2Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 3Department of Pediatrics and Microbiology, University of Texas Southwestern Medical Center A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes. Biology Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy Fei Liu1, Daniel J. Tschumperlin1 1Molecular and Integrative Physiological Sciences, Department of Environmental Health, Harvard School of Public Health The stiffness of the extracellular matrix strongly influences multiple behaviors of adherent cells. Matrix stiffness varies spatially throughout a tissue, and undergoes modification in various disease conditions. Here we develop methods to characterize spatial variations in stiffness in normal and fibrotic mouse lung tissue using atomic force microscopy microindentation. Medicine In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis Marcella A. Calfon1, Amir Rosenthal1,2, Georgios Mallas1,3, Adam Mauskapf1, R. Nika Nudelman2, Vasilis Ntziachristos2, Farouc A. Jaffer1 1Cardiovascular Research Center and Cardiology Division, Massachusetts General Hospital, Harvard Medical School, 2Institute for Biological and Medical Imaging, Helmholtz Zentrum München und Technische Universität München, 3Department of Electrical and Computer Engineering, Northeastern University We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata. Immunology and Infection Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging Jenny M. Tam1, Carlos E. Castro2, Robert J. W. Heath3, Michael K. Mansour1, Michael L. Cardenas1, Ramnik J. Xavier3, Matthew J. Lang4, Jatin M. Vyas1 1Department of Medicine, Division of Infectious Diseases, Massachusetts General Hospital, Harvard Medical School, 2Department of Mechanical and Aerospace Engineering, The Ohio State University, 3Center for Computational and Integrative Biology, Massachusetts General Hospital, Harvard Medical School, 4Dept. of Chemical and Biomolecular Engineering, Vanderbilt University A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells. Biology Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish Jessica S. Blackburn1, Sali Liu1, David M. Langenau2 1Department of Molecular Pathology, Massachusetts General Hospital, Harvard Medical School, 2Department of Molecular Pathology, Massachusetts General Hospital Cancer Center, Harvard Stem Cell Institute Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish. Biology Bioengineering Human Microvascular Networks in Immunodeficient Mice Ruei-Zeng Lin1, Juan M. Melero-Martin1 1Department of Cardiac Surgery, Children's Hospital Boston, Harvard Medical School Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature. Neuroscience Electrode Positioning and Montage in Transcranial Direct Current Stimulation Alexandre F. DaSilva1, Magdalena Sarah Volz2,3, Marom Bikson4, Felipe Fregni2 1Headache & Orofacial Pain Effort (H.O.P.E.), Biologic & Material Sciences, School of Dentistry, University of Michigan, 2Laboratory of Neuromodulation, Department of Physical Medicine & Rehabilitation, Spaulding Rehabilitation Hospital and Massachusetts General Hospital, Harvard Medical School, 3Charité, University Medicine Berlin, 4Department of Biomedical Engineering, The City College of New York Transcranial direct current stimulation (tDCS) is an established technique to modulate cortical excitability1,2. It has been used as an investigative tool in neuroscience due to its effects on cortical plasticity, easy operation, and safe profile. One area that tDCS has been showing encouraging results is pain alleviation 3-5. Immunology and Infection Seven Steps to Stellate Cells Patrick Maschmeyer1, Melanie Flach1, Florian Winau1 1Immune Disease Institute, Program in Cellular and Molecular Medicine at Children's Hospital, Department of Pathology, Harvard Medical School Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells. Neuroscience Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence Sava Sakadžić1, Emmanuel Roussakis2, Mohammad A. Yaseen1, Emiri T. Mandeville3, Vivek J. Srinivasan1, Ken Arai3, Svetlana Ruvinskaya1, Weicheng Wu1, Anna Devor1,4, Eng H. Lo3, Sergei A. Vinogradov2, David A. Boas1 1Optics Division, Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital and Harvard Medical School, 2Department of Biochemistry and Biophysics, University of Pennsylvania, 3Neuroprotection Research Laboratory, Departments of Radiology and Neurology, Massachusetts General Hospital and Harvard Medical School, 4Departments of Neurosciences and Radiology, University of California We present an experimental procedure for measuring the partial pressure of oxygen (pO2) in cerebral vasculature based on oxygen-dependent quenching of phosphorescence. Animal preparation and imaging procedures were outlined for both large field of view CCD-based imaging of pO2 in rats and 2-photon excitation based imaging of pO2 in mice. Immunology and Infection Competitive Homing Assays to Study Gut-tropic T Cell Migration Eduardo J. Villablanca1, J. Rodrigo Mora1 1Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School Competitive homing experiments allow to directly assessing the migratory properties of two different cell populations in a single mouse. Here we illustrate this procedure by comparing the migration of ex vivo-generated gut-tropic versus non-gut tropic T cells. Biology Primary Cell Cultures from Drosophila Gastrula Embryos Norbert Perrimon1,2, Jonathan Zirin1, Jianwu Bai1 1Department of Genetics, Harvard Medical School, 2Howard Hughes Medical Institute We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells. Medicine Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface Hilaire C. Lam1,2, Augustine M.K. Choi1, Stefan W. Ryter1 1Department of Medicine, Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital, Harvard Medical School, 2Cellular and Molecular Pathology, School of Medicine, University of Pittsburgh Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface as a model of differentiated respiratory epithelium. A protocol is described for isolating, culturing and exposing these cells to mainstream cigarette smoke, in order to study molecular responses to this environmental toxin. Bioengineering Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns Chia-Hua Lee1, Suman Bose2, Krystyn J. Van Vliet1, Jeffrey M. Karp3, Rohit Karnik2 1Department of Materials Science and Engineering, MIT - Massachusetts Institute of Technology, 2Department of Mechanical Engineering, MIT - Massachusetts Institute of Technology, 3HST Center for Biomedical Engineering and Harvard Stem Cell Institute, Brigham and Women's Hospital and Harvard Medical School We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis. Bioengineering Decellularization and Recellularization of Whole Livers Basak E. Uygun1, Gavrielle Price1, Nima Saeidi1, Maria-Louisa Izamis1, Tim Berendsen1, Martin Yarmush1, Korkut Uygun1 1Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Shriners Hospitals for Children Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique. Biology The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker Yue Zhang*1, Luv Kashyap*2, Annabel A. Ferguson2, Alfred L. Fisher2 1Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, 2Department of Medicine, Division of Geriatric Medicine and Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker. Medicine Intra-Operative Behavioral Tasks in Awake Humans Undergoing Deep Brain Stimulation Surgery John T. Gale*1,2, Clarissa Martinez-Rubio*1,2, Sameer A. Sheth1,2, Emad N. Eskandar1,2 1Nayef Al-Rodhan Laboratories for Cellular Neurosurgery and Neurosurgical Technology, Harvard Medical School, 2Department of Neurosurgery , Massachusetts General Hospital Deep brain stimulation surgery offers a unique opportunity to examine information encoding in the awake human brain. This article will describe intra-operative methods used to perform cognitive and behavioral tasks while simultaneously acquiring physiological data such as EMG, single-unit neuronal activity and/or local field potentials. Neuroscience In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons Heather Rice1, Seiyam Suth1, William Cavanaugh1, Jilin Bai2, Tracy L. Young-Pearse1 1Center for Neurologic Diseases, Brigham and Woman's Hospital and Harvard Medical School, 2Department of Physiology and Neurobiology, University of Connecticut In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration. Biology Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury Chiara Cianciolo Cosentino1, Beth L. Roman2, Iain A. Drummond3, Neil A. Hukriede1 1Department of Developmental Biology, University of Pittsburgh, 2Department of Biological Sciences, University of Pittsburgh, 3Department of Medicine and Genetics, Harvard Medical School We describe a technique of microinjecting the aminoglycoside, gentamicin, into 2 days post-fetilization (dpf) zebrafish larvae to induce acute kidney injury (AKI). We also describe a method for whole mount immunohistochemistry, plastic embedding and sectioning of zebrafish larvae to visualize the AKI mediated damage. Biology Hi-C: A Method to Study the Three-dimensional Architecture of Genomes. Nynke L. van Berkum*1, Erez Lieberman-Aiden*2,3,4,5, Louise Williams*2, Maxim Imakaev6, Andreas Gnirke2, Leonid A. Mirny3,6, Job Dekker1, Eric S. Lander2,7,8 1Program in Gene Function and Expression, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 2Broad Institute of Harvard and Massachusetts Institute of Technology, 3Division of Health Sciences and Technology, Massachusetts Institute of Technology, 4Program for Evolutionary Dynamics, Department of Organismic and Evolutionary Biology, Department of Mathematics, Harvard University, 5Department of Applied Mathematics, Harvard University, 6Department of Physics, Massachusetts Institute of Technology, 7Department of Systems Biology, Harvard Medical School, 8Department of Biology, Massachusetts Institute of Technology The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale. Neuroscience Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning Valeria Righi1,2,3, Yiorgos Apidianakis2,4, Laurence G. Rahme2,4, A. Aria Tzika1,2,3 1NMR Surgical Laboratory, Department of Surgery, Massachusetts General Hospital, Harvard Medical School, 2Shriners Burn Institute, 3Department of Radiology, Athinoula A. Martinos Center of Biomedical Imaging, Harvard Medical School, 4Molecular Surgery Laboratory, Department of Surgery, Massachusetts General Hospital, Harvard Medical School This technique enables the use of high-resolution magic angle spinning proton MR spectroscopy (HRMAS 1H-MRS) for molecular characterization of live Drosophila melanogaster with a conventional 14.1 tesla spectrometer equipped with an HRMAS probe. Biology Monitoring Acupuncture Effects on Human Brain by fMRI Kathleen K. S. Hui1, Vitaly Napadow1, Jing Liu1, Ming Li1, Ovidiu Marina1,2, Erika E. Nixon1, Joshua D. Claunch1, Lauren LaCount1, Tara Sporko1, Kenneth K. Kwong1 1Department of Radiology, Massachusetts General Hospital and Harvard Medical School, 2William Beaumont Hospital FMRI and physiological monitoring is used to study the effects of Acupuncture on the central and peripheral nervous systems. Acupuncture mobilizes a limbic-paralimbic-neocortical network, with great overlap with the default mode network, to modulate neurological activity, possibly related to its autonomic effect in the peripheral nervous system. Biology Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay Arkadiusz W. Kulczyk1, Nathan A. Tanner1, Joseph J. Loparo1, Charles C. Richardson1, Antoine M. van Oijen1 1Harvard Medical School We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Biology Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. Mark Parker1,2,3, Aurore Brugeaud1,2, Albert S. B. Edge1,2,4 1Department of Otology and Laryngology, Harvard Medical School, 2Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, 3Department of Communication Sciences and Disorders, Emerson College, 4Program in Speech and Hearing Bioscience and Technology, Division of Health Science and Technology, Harvard This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation. Biology PuraMatrix Encapsulation of Cancer Cells Adnan O. Abu-Yousif1, Imran Rizvi1,2, Conor L. Evans1, Jonathan P. Celli1, Tayyaba Hasan1,3 1Wellman Center for Photomedicine Massachusetts General Hospital, Harvard Medical School, 2Thayer School of Engineering, Dartmouth College, 3Department of Dermatology, Harvard Medical School This video demonstrates how to encapsulate and culture cancer cells in PuraMatrix, a commercially available self assembling peptide gel. Biology Retro-orbital Injection in Adult Zebrafish Emily K. Pugach1,2, Pulin Li1,2, Richard White1,2,3, Leonard Zon1,2 1Department of Hematology and Oncology, Children’s Hospital Boston, 2Harvard Stem Cell Institute, Harvard Medical School, 3Department of Medical Oncology, Dana Farber Cancer Institute Here we show how to do retro-orbital injection in adult zebrafish. Biology Visualizing Single-molecule DNA Replication with Fluorescence Microscopy Nathan A. Tanner1, Joseph J. Loparo1, Antoine M. van Oijen1 1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time. Biology Method for Measurement of Viral Fusion Kinetics at the Single Particle Level Daniel L. Floyd1, Stephen C. Harrison1,2, Antoine M. van Oijen1 1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 2Howard Hughes Medical Institute, Harvard Medical School We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation. Biology Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure Kenneth K. Kwong1,2, Lenuta Kloetzer1,2,3,4, Kelvin K. Wong5,6, Jia-Qian Ren1,2, Braden Kuo1,2,3,4, Yan Jiang7, Y. Iris Chen1,2, Suk-Tak Chan1,2,8, Geoffrey S. Young9, Stephen T.C. Wong5,6 1Department of Radiology, Massachusetts General Hospital, Harvard Medical School, 2Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital, Harvard Medical School, 3Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School, 4Department of Medicine, Massachusetts General Hospital, Harvard Medical School, 5Center for biotechnology and Informatics, The Methodist Hospital Research Institute, 6Department of Radiology, The Methodist Hospital, Weill Cornell Medical College, 7Bejing University of Chinese Medicine, 8Department of Health Technology and Informatics, The Hong Kong Polytechnic University, 9Department of Radiology, Brigham and Women's Hospital, Harvard Medical School Gua Sha, traditional Chinese therapeutic skin scraping, causes subcutaneous microvascular blood extravasation. We report a protocol of bioluminescence imaging of HO-1-luciferase transgenic mice to demonstrate that Gua Sha upregulates heme oxygenase-1 (HO-1) in multiple organs. Biology Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila Claudio Vinegoni1, Daniel Razansky2, Chrysoula Pitsouli3, Norbert Perrimon3, Vasilis Ntziachristos2, Ralph Weissleder1 1Center for Systems Biology, Massachusetts General Hospital, 2Institute for Biological and Medical Imaging (IBMI), Technical University of Munich and Helmholtz Center Munich, 3Department of Genetics, Harvard Medical School and Howard Hughes Medical Institute Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster. Biology Optical Mapping of Langendorff-perfused Rat Hearts Bjoern Sill1, Peter E. Hammer1,2, Douglas B. Cowan1 1Department of Anesthesiology, Perioperative and Pain Medicine, Children's Hospital Boston and Harvard Medical School, 2Departments of Cardiac Surgery, Children's Hospital Boston and Harvard Medical School This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS). Biology Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue Charmaine Y. Pietersen1, Maribel P. Lim1, Tsung-Ung W. Woo1,2,3 1Department of Structural and Molecular Neuroscience, McLean Hospital, 2Department of Psychiatry, Harvard Medical School, 3Department of Psychiatry, Beth Israel Deaconess Medical Center We describe a process using laser-capture microdissection to isolate and extract RNA from a homogeneous cell population, pyramidal neurons, in layer III of the superior temporal gyrus in postmortem human brains. We subsequently linearly amplify (T7-based) mRNA, and hybridize the sample to the Affymetrix human X3P microarray. Biology Psychophysiological Stress Assessment Using Biofeedback Inna Khazan1,2 1Behavioral Medicine, Cambridge Health Alliance, 2Harvard Medical School In this video we will describe one method of assessing person's psychophysiological reaction to stress using biofeedback. We will also present general guidelines for treatment planning. Biology Making MR Imaging Child's Play - Pediatric Neuroimaging Protocol, Guidelines and Procedure Nora M. Raschle1,2, Michelle Lee1, Roman Buechler1, Joanna A. Christodoulou3, Maria Chang1, Monica Vakil1, Patrice L. Stering1, Nadine Gaab1,3,4 1Department of Developmental Medicine, Children’s Hospital Boston, 2Department of Neuropsychology, University of Zurich, 3Graduate School of Education, Harvard, 4Harvard Medical School Despite an increase in the use of structural and functional magnetic resonance imaging (fMRI) in humans, the study of young pediatric populations remains a challenge. We present a hands-on, step-by-step video protocol including guidelines for clinicians and researchers intending to perform (f)MRI in young children. Biology Implantation of Engineered Tissue in the Rat Heart Bjoern Sill1, Ivan V. Alpatov2, Christina A. Pacak2, Douglas B. Cowan2 1Department of Anesthesiology, Perioperative and Pain Medicine, Children's Hospital Boston and Harvard Medical School, 2Department of Anesthesiology, Perioperative and Pain Medicine, Children’s Hospital Boston Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat. Biology Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging Claudio Vinegoni1,2, Daniel Razansky3, Jose-Luiz Figueiredo1,2, Lyuba Fexon1,2, Misha Pivovarov1,2, Matthias Nahrendorf1,2, Vasilis Ntziachristos3, Ralph Weissleder1,2 1Center for Systems Biology, Harvard Medical School, 2Center for Systems Biology, MGH - Massachusetts General Hospital, 3Institute for Biological and Medical Imaging, Technical University of Munich and Helmholtz Center Munich We suggest a Born normalized approach for Optical Projection Tomography (BnOPT) that accounts for the absorption properties of imaged samples to obtain accurate and quantitative fluorescence tomographic reconstructions. We use the proposed algorithm to reconstruct the fluorescence molecular probe distribution within small animal organs. Biology Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants Yan Zhang 1,2, Kyle McCulloch2, Jarema Malicki2 1The Second Teaching Hospital of Jilin University, 2Department of Ophthalmology, Harvard Medical School Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens. Biology Fabrication of Myogenic Engineered Tissue Constructs Christina A. Pacak1,2, Douglas B. Cowan1,2 1Department of Anesthesiology, Children's Hospital Boston and Harvard Medical School, 2Perioperative and Pain Medicine, Children's Hospital Boston and Harvard Medical School Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1]. Biology Microinjection of Zebrafish Embryos to Analyze Gene Function Jonathan N. Rosen1,2, Michael F. Sweeney1, John D. Mably1,2 1Department of Genetics, Harvard Medical School, 2Department of Cardiology, Children’s Hospital Boston This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development. Biology Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium Georg Wiese1, Steven R. Barthel2, Charles J. Dimitroff2 1Department of Dermatology, Brigham and Women's Hospital, 2Department of Dermatology, Brigham and Women's Hospital and Harvard Medical School This report provides a visual depiction of parallel-plate flow chamber analysis for studying leukocyte endothelial interactions under physiologic shear stress. This method is particularly useful for investigating the role of endothelial (E)-selectin and leukocyte E-selectin ligands that trigger leukocyte rolling on endothelial cell surfaces. Biology Olfactory Behavioral Testing in the Adult Mouse Rochelle M. Witt1,2, Meghan M. Galligan1,2, Jennifer R. Despinoy1,2, Rosalind Segal1,2 1Department of Pediatric Oncology, Dana Farber Cancer Institute, 2Department of Neurobiology, Harvard Medical School Fundamental, yet unique properties of the rodent olfactory system have led to its increasing study among biologists. A relatively simple assessment of its function is then also needed. Here we describe sensitive tests for the characterization of mouse olfactory sensitivity and preference. Biology Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons Hae Young Lee1, Lloyd A. Greene2, Carol A. Mason2,3, M. Chiara Manzini2,4 1Department of Genetics and Development, Columbia University, 2Department of Pathology and Cell Biology, Columbia University, 3Department of Neuroscience, Columbia University, 4Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Medical School Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse. Biology Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures Maria F. Pazyra-Murphy1,2, Rosalind A. Segal1,2 1Department of Pediatric Oncology, Dana Farber Cancer Institute, 2Department of Neurobiology, Harvard Medical School Here we describe the technique of preparing and maintaining compartmented chambers for culturing sensory neurons of the dorsal root ganglia. Biology A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities Farida Emran1, Jason Rihel1, John E. Dowling1 1Department of Molecular and Cell Biology, Harvard We developed the Visual-Motor Response to quantitate the motor output of larval zebrafish in response to light increments and decrements. We also examined zebrafish vision mutants, including the no optokinetic response (nrc) mutants, which were thought to be completely blind when tested by another vision assay, the optokinetic reflex. Biology Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay Shaochong Zhang1, Axel Nohturfft1,2 1Department of Molecular and Cell Biology, Harvard, 2Molecular and Metabolic Signalling Centre, Division of Basic Medical Sciences, St. George's University of London Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega. Biology Construction and Implantation of a Microinfusion System for Sustained Delivery of Neuroactive Agents. Miles G. Cunningham1, Ryan P. O'Connor1, Sydney E. Wong1 1Harvard Medical School As neuroscience inquiry becomes more sophisticated, investigation of brain structures and circuitry requires improved levels of accuracy and higher resolution. We have developed a method for the preparation and implantation of a chronic infusion system within the brain utilizing a borosilicate microcannula with a tip diameter of 50 microns. Biology Derivation of Human Embryonic Stem Cells by Immunosurgery Alice E. Chen1, Douglas A. Melton1 1Department of Molecular and Cell Biology, Harvard The ability of human embryonic stem cells to self-renew and differentiate into all cell types of the body suggests that they hold great promise for both medical applications and as a research tool for addressing fundamental questions in development and disease. Here, we provide a concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos by immunosurgical isolation of the inner cell mass. Biology Title Cell Encapsulation by Droplets Sangjun Moon1,2, Pei-Ann Lin1,2, Hasan Onur Keles1,2, Seung-Schick Yoo3, Utkan Demirci1,2,4 1Bio-Acoustic-MEMS Laboratory in Medicine (BAMM), HST-Center for Bioengineering, Brigham and Women's, Harvard Medical School, 2Bio-Acoustic-MEMS Laboratory in Medicine (BAMM), HST-Center for Bioengineering, Brigham and Women's Hospital, 3Brigham and Women's Hospital, Harvard Medical School, 4Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology; Center for Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital Biology Biology of Microbial Communities - Interview Roberto Kolter1 1Department of Microbiology and Molecular Genetics, Harvard Medical School Biology In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy Akihiro Asai1, Nita Sahani1, Yasuyoshi Ouchi2, Jeevendra Martyn1, Shingo Yasuhara1 1Department of Anesthesiology and Critical Care, Shriners Hospital for Children, Massachusetts General Hospital, and Harvard Medical School, 2Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions. Biology Ole Isacson: Development of New Therapies for Parkinson's Disease Ole Isacson1 1McLean Hospital, Harvard Stem Cell Institute, Harvard Medical School Ole Isacson gives a concise overview of Parkinsons's disease, its causes, therapeutic strategies, and advances in Parkinson's research. Biology Thin Sectioning of Slice Preparations for Immunohistochemistry Jae-Joon Park1,2, Miles G. Cunningham2 1Department of Medicine, Yonsei University College of Medicine, Severance Hospital, 2Department of Psychiatry, Harvard Medical School The present method allows reproducible cryostat sectioning of small, difficult-to-manage, tissue pieces, such as biopsies and brain slices. We utilize a simple aluminum freezing stage to facilitate handling of tissue and a standard cryostat to routinely produce 5-10 micron serial sections from 400 micron thick brain slices. Biology Studying Aggression in Drosophila (fruit flies) Sibu Mundiyanapurath1, Sarah Certel1, Edward A. Kravitz1 1Department of Neurobiology, Harvard Medical School Editorial Interview with Edward Kravitz Edward A. Kravitz1 1Department of Neurobiology, Harvard Medical School Biology Isolation and Transplantation of Hematopoietic Stem Cells (HSCs) Cristina Lo Celso1, David Scadden1 1Center for Regenerative Medicine, Massachusetts General Hospital, Harvard Stem Cell Institute, Harvard Medical School Biology Transplantation of Whole Kidney Marrow in Adult Zebrafish Jocelyn LeBlanc1, Teresa Venezia Bowman1, Leonard Zon1 1Children's Hospital, Harvard Stem Cell Institute, Harvard Medical School In this article, we demonstrate a method to perform HCT in adult zebrafish. Biology Dissection of 6.5 dpc Mouse Embryos Kelly Shea1, Niels Geijsen1 1Massachusetts General Hospital, Harvard Stem Cell Institute, Harvard Medical School Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]). Biology Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells Shannon McKinney-Freeman1, George Daley1 1Childrens Hospital, Harvard Stem Cell Institute, Harvard Medical School This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines. Biology Tracheotomy: A Method for Transplantation of Stem Cells to the Lung Yakov Peter1 1Dept. of Cell Biology, Harvard Medical School Significant breakthroughs in stem cell identification are continuously being made. To translate these discoveries, however, novel methods for cellular delivery must be devised. Here I report that the airways provide a safe route for stem cell transplantation to the lungs. Biology Denaturing Gradient Gel Electrophoresis (DGGE) Celeste Peterson1 1Department of Microbiology and Molecular Genetics, Harvard Medical School Biology Nuclear Transfer into Mouse Oocytes Dieter Egli1, Kevin Eggan1 1Department of Molecular and Cell Biology, Harvard This movie and the protocol are intended to help learning nuclear transfer. Biology Measuring Exocytosis in Neurons Using FM Labeling Jamila Newton1, Venkatesh Murthy1 1Department of Molecular and Cell Biology, Harvard The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with the red fluorescent dye FM 4-64 to measure the rate of presynaptic vesicle release in hippocampal neuronal cultures. Biology ES Cell-derived Neuroepithelial Cell Cultures Shreeya Karki1, Jan Pruszak1, Ole Isacson1, Kai C Sonntag1 1McLean Hospital, Harvard Medical School Derivation of neuroepithelial precursors from embryonic stem (ES) cells using stromal cell-derived inducing activity (SDIA). Biology Long-term Imaging Mammalian Cells using Wide-Field Microscopy Meg Bentley1, Randy King1 1Dept. of Cell Biology, Harvard Medical School Biology Horizontal Slice Preparation of the Retina Ryosuke Enoki1, Tatjana C. Jakobs2, Amane Koizumi2 1Dpt of Physiology and Biophysics, Dalhousie University, 2Massachusetts General Hospital, Harvard Medical School Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact. Biology Human ES cells: Starting Culture from Frozen Cells Erin Trish1, John Dimos1, Kevin Eggan1 1Department of Molecular and Cell Biology, Harvard Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock. Biology Monitoring Actin Disassembly with Time-lapse Microscopy Hao Yuan Kueh1 1Dept. of Systems Biology, Harvard Medical School Biology Passaging HuES Human Embryonic Stem Cell-lines with Trypsin. Erin Trish1, John Dimos1, Kevin Eggan1 1Department of Molecular and Cell Biology, Harvard In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Brought to you by JoVE. Biology Freezing Human ES Cells Erin Trish1, John Dimos1, Kevin Eggan1 1Department of Molecular and Cell Biology, Harvard Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.
