Rutgers University View Institution's Website 46 articles published in JoVE Immunology and Infection Enrichment of Native and Recombinant Extracellular Vesicles of Mycobacteria Praapti Jayaswal1, Mohd Ilyas1, Kuljit Singh1,2, Saurabh Kumar1,3, Lovely Sisodiya1, Sapna Jain1, Rahul Mahlawat1, Nishant Sharma1,4, Vishal Gupta1, Krishnamohan Atmakuri1 1Bacterial Pathogenesis Laboratory, Infectious Diseases and Immunology Group, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, 2Clinical Microbiology Division, CSIR-Indian Institute of Integrative Medicine, 3ICAR-Research Complex for Eastern Region, 4Public Health Research Institute, Rutgers University This protocol details the enrichment of native mycobacterial extracellular vesicles (mEVs) from axenic cultures of Mycobacterium smegmatis (Msm) and how mCherry (a red fluorescent reporter)-containing recombinant MsmEVs can be designed and enriched. Lastly, it verifies the novel approach with the enrichment of MsmEVs containing the EsxA protein of Mycobacterium tuberculosis. Neuroscience Isolation of Targeted Hypothalamic Neurons for Studies of Hormonal, Metabolic, and Electrical Regulation Kathera-Ibarra-Forzisi Elena1, Christian Roser1, Anika Gaur1, Federico Sesti1 1Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, Rutgers University Here we present a protocol to grow specific hypothalamic cell subtypes in culture. The cells can be selected based on opportune/unique membrane markers and used in many applications, including immunofluorescence, electrophysiological, and biochemical assays. Biology Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells Qin He1, Huiliang Zhang2, Xingyun Qi1 1Department of Biology, Rutgers University, 2Pharmacology and Toxicology Department, University of Arkansas for Medical Sciences Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup. Bioengineering Multi-Stream Perfusion Bioreactor Integrated with Outlet Fractionation for Dynamic Cell Culture Patrick Erickson1, Aneesha Doshi2, Gunjan Jetley2, Param Amin2, Aamena Mejevdiwala2, Ashna Patel2, Raphaela Bento2, Biju Parekkadan2,3 1Department of Chemical and Biochemical Engineering, Rutgers University, 2Department of Biomedical Engineering, Rutgers University, 3Department of Medicine, Rutgers Biomedical Health Sciences This paper presents a method to construct and operate a low-cost, multichannel perfusion cell culture system for measuring the dynamics of secretion and absorption rates of solutes in cellular processes. The system can also expose cells to dynamic stimulus profiles. Chemistry Dose Uptake of Platinum- and Ruthenium-based Compound Exposure in Zebrafish by Inductively Coupled Plasma Mass Spectrometry with Broader Applications Brittany F. Karas1,2, Cathleen L. Doherty1, Kristin R. Terez2, Leonor Côrte-Real3, Keith R. Cooper1,2, Brian T. Buckley1 1Environmental and Occupational Health Sciences Institute, Rutgers University, 2Department of Biochemistry and Microbiology, Rutgers University, 3Centro de Química Estrutural and Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa The increased rate of pharmaco- and toxicokinetic analyses of metals and metal-based compounds in zebrafish can be advantageous for environmental and clinical translation studies. The limitation of unknown waterborne exposure uptake was overcome by conducting trace metal analysis on digested zebrafish tissue using inductively coupled plasma mass spectrometry. Cancer Research Natural Killer (NK) and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line Minh Ma1,2, Saiaditya Badeti1, James K. Kim1, Dongfang Liu1,3 1Department of Pathology, Immunology and Laboratory Medicine, Rutgers-New Jersey Medical School, 2Department of Microbiology, Biochemistry & Molecular Genetics, Public Health Research Institute Center, New Jersey Medical School, Rutgers University, 3Center for Immunity and Inflammation, New Jersey Medical School, Rutgers-The State University of New Jersey Here, we present a method to expand peripheral blood natural killer (PBNK), NK cells from liver tissues, and chimeric antigen receptor (CAR)-NK cells derived from peripheral blood mononuclear cells (PBMCs) or cord blood (CB). This protocol demonstrates the expansion of NK and CAR-NK cells using 221-mIL-21 feeder cells in addition to the optimized purity of expanded NK cells. Biochemistry A Robust Single-Particle Cryo-Electron Microscopy (cryo-EM) Processing Workflow with cryoSPARC, RELION, and Scipion Megan C. DiIorio1, Arkadiusz W. Kulczyk1,2 1Institute for Quantitative Biomedicine, Rutgers University, 2Department of Biochemistry & Microbiology, Rutgers University This article describes how to effectively utilize three cryo-EM processing platforms, i.e., cryoSPARC v3, RELION-3, and Scipion 3, to create a single and robust workflow applicable to a variety of single-particle data sets for high-resolution structure determination. Neuroscience Using Optogenetics to Reverse Neuroplasticity and Inhibit Cocaine Seeking in Rats Matthew T. Rich1,2, Yanhua H. Huang1, Mary M. Torregrossa1 1Department of Psychiatry, University of Pittsburgh, 2Department of Psychiatry, Rutgers University The methods described here outline a procedure used to optogenetically reverse cocaine-induced plasticity in a behaviorally-relevant circuit in rats. Sustained low-frequency optical stimulation of thalamo-amygdala synapses induces long-term depression (LTD). In vivo optogenetically-induced LTD in cocaine-experienced rats resulted in the subsequent attenuation of cue-motivated drug seeking. Behavior Real-Time Proxy-Control of Re-Parameterized Peripheral Signals using a Close-Loop Interface Vilelmini Kalampratsidou1,2, Steven Kemper3, Elizabeth B. Torres1,2,4,5,6 1Center for Cognitive Science, Rutgers University, 2Department of Computer Science, Rutgers University, 3Music Department, Mason Gross School of the Arts, Rutgers University, 4Psychology Department, Rutgers University, 5Sensory Motor Integration Lab, Rutgers University, 6Computational Biomedicine Imaging and Modelling Center, Rutgers University We present protocols and methods of analyses to build co-adaptive interfaces that stream, parameterize, analyze, and modify human body and heart signals in close-loop. This setup interfaces signals derived from the peripheral and central nervous systems of the person with external sensory inputs to help track biophysical change. Neuroscience Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans Meghan Lee Arnold*1, Jason Cooper*1, Barth D. Grant1, Monica Driscoll1 1Department of Molecular Biology and Biochemistry, Rutgers University This protocol describes approaches for detection and quantitation of large aggregate and/or organelle extrusions (~4 µm) produced by C. elegans cells in the form of membrane-bound exophers. We describe strains, growth conditions, scoring criteria, timing, and microscopy considerations needed to facilitate dissection of this debris expulsion mechanism. Developmental Biology RiboTag Immunoprecipitation in the Germ Cells of the Male Mouse Lauren G. Chukrallah1, Kelly Seltzer1, Elizabeth M. Snyder1 1Department of Animal Science, Rutgers University Here, we describe the immunoprecipitation of ribosomes and associated RNA from select populations of adult male mouse germ cells using the RiboTag system. Strategic breeding and careful immunoprecipitation result in clean, reproducible results that inform on the germ cell translatome and provide insight into the mechanisms of mutant phenotypes. Biochemistry Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription Malhar Desai1,2, Rong Di3, Huizhou Fan1,2 1Department of Pharmacology, Robert Wood Johnson Medical School, Rutgers University, 2Graduate Program in Physiology and Integrative Biology, School of Graduate Studies, Rutgers University, 3Department of Plant Biology, School of Environmental and Biological, Rutgers University Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative. Behavior Dynamic Digital Biomarkers of Motor and Cognitive Function in Parkinson's Disease Jihye Ryu1,2, Joe Vero1, Roseanne D. Dobkin3, Elizabeth B. Torres1,2,4 1Department of Psychology, Rutgers University, 2Rutgers University Center for Cognitive Science, Rutgers University, 3Rutgers-Robert Wood Johnson Medical School, Department of Psychiatry, Rutgers University, 4Center for Biomedicine, Imaging and Modelling, Department of Computer Science, Rutgers University This protocol offers a digitization of portions of traditional clinical tasks commonly used to measure cognition and motor control in Parkinson’s disease. Clinical tasks are digitized while biophysical rhythms are co-registered from different functional levels of the nervous systems, ranging from voluntary, spontaneous, automatic to autonomic. Developmental Biology Ex Vivo Perfusion of the Rodent Placenta Jeanine N. D'Errico1, Sara B. Fournier2, Phoebe A. Stapleton1,2 1Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, 2Environmental and Occupational Health Sciences Institute Here is presented a protocol of ex vivo maternal-fetal vascular perfusion to enable the administration of a test article into maternal vasculature and to evaluate placental transfer of xenobiotic particles or pharmacological agents in addition to alterations in placental physiology. Environment Colletotrichum fioriniae Development in Water and Chloroform-based Blueberry and Cranberry Floral Extracts Timothy J. Waller1, Joshua D. Gager1, Peter V. Oudemans1 1Department of Plant Biology, P. E. Marucci Center for Blueberry and Cranberry Research and Extension, Rutgers University Here, bioassays designed to monitor the development of a fungal pathogen, Colletotrichum fioriniae, in the presence of blueberry or cranberry floral extracts on glass coverslips are described. Water-, chloroform-, and field rainwater- based floral extraction techniques are detailed as well as insight into how this information can be applied. Bioengineering Mouse Model of Pressure Ulcers After Spinal Cord Injury Suneel Kumar1, Yuying Tan1, Martin L. Yarmush1,2, Biraja C. Dash3, Henry C. Hsia3, Francois Berthiaume1 1Department of Biomedical Engineering, Rutgers University, 2Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Shriners Hospitals for Children, 3Department of Surgery, Yale School of Medicine, Yale University Here, we describe a simple method to induce clinically relevant skin pressure ulcers (PUs) in a mouse model of spinal cord injury (SCI). This model can be used in pre-clinical studies to screen for different therapeutics for healing PUs in SCI patients. Immunology and Infection Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay Almin I. Lalani1, Sining Zhu1, Ping Xie2,3 1Department of Cell Biology and Neuroscience and Graduate Program in Cellular and Molecular Pharmacology, Rutgers University, 2Department of Cell Biology and Neuroscience, Rutgers University, 3Rutgers Cancer Institute of New Jersey In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization. Behavior A Networked Desktop Virtual Reality Setup for Decision Science and Navigation Experiments with Multiple Participants Hantao Zhao1, Tyler Thrash1,2,3, Stefan Wehrli4, Christoph Hölscher1, Mubbasir Kapadia5, Jascha Grübel1, Raphael P. Weibel1, Victor R. Schinazi1 1Chair of Cognitive Science, ETH Zürich, 2Digital Society Initiative, University of Zürich, 3Department of Geography, University of Zürich, 4Decision Science Laboratory, ETH Zürich, 5Computer Science Department, Rutgers University This paper describes a method for conducting multi-user experiments on decision-making and navigation using a networked computer laboratory. Cancer Research Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer Larry C. Cheng*1,2, Zhen Li*3, Thomas G. Graeber4, Nicholas A. Graham5, Justin M. Drake1,2,3,6,7 1Graduate Program in Cellular and Molecular Pharmacology, School of Graduate Studies, Rutgers University, The State University of New Jersey, 2Graduate Program in Quantitative Biomedicine, School of Graduate Studies, Rutgers University, The State University of New Jersey, 3Department of Medicine, Division of Medical Oncology, Rutgers Robert Wood Johnson Medical School, 4Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, Jonsson Comprehensive Cancer Center, UCLA Metabolomics Center, and California NanoSystems Institute, David Geffen School of Medicine, University of California, Los Angeles, 5Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, 6Pharmacology, Rutgers Robert Wood Johnson Medical School, 7Cancer Metabolism and Growth Program, Rutgers Cancer Institute of New Jersey This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based proteomics. Medicine Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry (LCM-LC/MS) Matthew Zimmerman1, Landry Blanc1, Pei-Yu Chen1, Véronique Dartois1, Brendan Prideaux1 1Public Health Research Institute, New Jersey Medical School, Rutgers, The State University of New Jersey Here, we describe a protocol using laser capture microdissection coupled with LC/MS analysis to spatially-quantify drug distributions within pulmonary tuberculosis granulomas. The approach has broad applicability to quantifying drug concentrations within tissues at high spatial detail. Genetics Using Mouse Oocytes to Assess Human Gene Function During Meiosis I Diego Marin*1,2, Alexandra L. Nguyen*3, Richard T. Scott, Jr.1,2, Karen Schindler3 1IVI-RMA New Jersey, 2Jefferson College of Biomedical Sciences, Thomas Jefferson University, 3Department of Genetics, Rutgers, The State University of New Jersey As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes. Developmental Biology Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs) Madeline Williams*1, Smrithi Prem*1, Xiaofeng Zhou1, Paul Matteson2, Percy Luk Yeung3, Chi-Wei Lu3, Zhiping Pang4, Linda Brzustowicz5, James H. Millonig2, Emanuel Dicicco-Bloom1 1Department of Neuroscience and Cell Biology, Rutgers Robert Wood Johnson Medical School, 2Center for Advanced Biotechnology and Medicine, Department of Neuroscience and Cell Biology, Rutgers Robert Wood Johnson Medical School, 3The Child Health Institute of NJ, Department of Obstetrics, Gynecology, and Reproductive Services, Rutgers Robert Wood Johnson Medical School, 4The Child Health Institute of NJ, Department of Neuroscience and Cell Biology, Rutgers Robert Wood Johnson Medical School, 5Department of Genetics, Rutgers University Neurodevelopmental processes such as proliferation, migration, and neurite outgrowth are often perturbed in neuropsychiatric diseases. Thus, we present protocols to rapidly and reproducibly assess these neurodevelopmental processes in human iPSC-derived NPCs. These protocols also allow the assessment of the effects of relevant growth factors and therapeutics on NPC development. Genetics In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells Mingzhu Fang1, Hwan-Goo Kang2, Youngil Park2, Brian Estrella1, Helmut Zarbl1 1Department of Environmental and Occupational Health, School of Public Health, NIEHS Center for Environmental Exposures and Disease, Environmental and Occupational Health Sciences Institute, Rutgers University, 2Veterinary Drugs & Biologics Division, Animal and Plant Quarantine Agency An in vitro bioluminescence assay to determine cellular circadian rhythm in mammary epithelial cells is presented. This method utilizes mammalian cell reporter plasmids expressing destabilized luciferase under the control of the PERIOD 2 gene promoter. It can be adapted to other cell types to evaluate organ-specific effects on circadian rhythm. Immunology and Infection An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions Jansy P. Sarathy1, Hsin-pin Ho Liang1, Danielle Weiner2, Jacqueline Gonzales2, Laura E. Via2, Véronique Dartois1 1Public Health Research Institute Centre, New Jersey Medical School, Rutgers, 2Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, NIAID, NIH Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum. Bioengineering A Versatile Method of Patterning Proteins and Cells Anil B. Shrirao*1, Frank H. Kung*2, Derek Yip3, Bonnie L. Firestein2, Cheul H, Cho3, Ellen Townes-Anderson4 1Department of Biomedical Engineering, Rutgers University, 2Department of Cell Biology and Neuroscience, Rutgers University, 3Department of Biomedical Engineering, New Jersey Institute of Technology, 4Department of Pharmacology, Physiology, and Neuroscience, Rutgers New Jersey Medical School This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research. Neuroscience Automated Analysis of C. elegans Swim Behavior Using CeleST Software Carolina Ibáñez-Ventoso1, Christopher Herrera2, Esteban Chen1, Douglas Motto2, Monica Driscoll1 1Department of Molecular Biology and Biochemistry, Rutgers University, 2Computing Research and Education Building (CoRE), Rutgers University An efficient and simple methodology for computer-based analysis of nematode swimming behavior in liquid is described. The method requires little to no investment for C. elegans laboratories. The hardware used is standard, and the computer software for the behavioral analysis (CeleST) is an open source one. Biology Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes Sarah J. Radford1, Kim S. McKim1,2 1Waksman Institute, Rutgers University, 2Department of Genetics, Rutgers University We present protocols for the collection, preparation, and imaging of mature Drosophila oocytes. These methods allow the visualization of chromosome behavior and spindle assembly and function during meiosis. Cancer Research A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication Jason D. Domogauer1, Sonia M. de Toledo1, Edouard I. Azzam1 1Department of Radiology, New Jersey Medical School, Rutgers University We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions. Neuroscience Quantitative Measurement of Relative Retinoic Acid Levels in E8.5 Embryos and Neurosphere Cultures Using the F9 RARE-Lacz Cell-based Reporter Assay Myka R. Ababon*1, Bo I. Li*2, Paul G. Matteson1, James H. Millonig1,2 1Center for Advanced Biotechnology and Medicine, Rutgers University, 2Department of Neuroscience and Cell Biology, Rutgers University Methods to accurately measure retinoic acid (RA) levels in small amounts of tissue do not exist. This protocol describes the easy, quantitative measurement of relative RA levels in E8.5 embryos and neurospheres using an RA reporter cell line. Developmental Biology Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells Jadali Azadeh1, Zhichao Song*1, Alejandra S. Laureano*1, Alana Toro-Ramos1, Kelvin Kwan1 1Cell Biology & Neuroscience, Rutgers University The current protocols to maintain immortalized multipotent otic progenitor (iMOP) cells and otic differentiation are described. Culture conditions and molecular markers that indicate differentiation into sensory epithelia and spiral ganglion neurons (SGN) are highlighted. Behavior Infant Auditory Processing and Event-related Brain Oscillations Gabriella Musacchia1,2,3, Silvia Ortiz-Mantilla1, Teresa Realpe-Bonilla1, Cynthia P. Roesler1, April A. Benasich1 1Center for Molecular & Behavioral Neuroscience, Rutgers University, State University of New Jersey, Newark, 2Department of Audiology, University of the Pacific, 3Department of Otolaryngology, Head & Neck Surgery, Stanford University High-density electroencephalography (dEEG) is being used increasingly to study brain development and plasticity in the early years of life. Here we present an application of sophisticated analysis techniques that builds on traditional EEG recording to understand the oscillatory dynamics of rapid auditory processing in the infant brain. Chemistry Electrochemically and Bioelectrochemically Induced Ammonium Recovery Sylvia Gildemyn1, Amanda K. Luther2, Stephen J. Andersen1, Joachim Desloover1, Korneel Rabaey1 1Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, 2Department of Environmental Sciences, Rutgers University We demonstrate the extraction of ammonium from an ammonium-rich stream using an electrochemical and a bioelectrochemical system. The reactor setup, operation and data analysis are discussed. Behavior Measuring Attentional Biases for Threat in Children and Adults Vanessa LoBue1 1Department of Psychology, Rutgers University Here we describe a touch-screen visual search paradigm that can be used to study threat detection across the lifespan. The paradigm has already been used in various studies demonstrating that both children and adults detect threatening stimuli like snakes, spiders, and angry faces faster than non-threatening stimuli. Biology Preparation of DNA-crosslinked Polyacrylamide Hydrogels Michelle L. Previtera1, Noshir A. Langrana2,3 1New Jersey Neuroscience Institute, JFK Medical Center, 2Department of Biomedical Engineering, Rutgers University, 3Department of Mechanical and Aerospace Engineering, Rutgers University Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels. Biology High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately Wenjin Chen2,3, Chung Wong1,3, Evan Vosburgh1,3, Arnold J. Levine3,4, David J. Foran2,3, Eugenia Y. Xu1,3 1Raymond and Beverly Sackler Foundation, New Jersey, 2Histopathology and Imaging Shared Resource, Rutgers University, 3Rutgers Cancer Institute of New Jersey, Rutgers University, 4School of Natural Sciences, Institute for Advanced Study, New Jersey We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens. Behavior Methods to Explore the Influence of Top-down Visual Processes on Motor Behavior Jillian Nguyen1, Thomas V. Papathomas2,3, Jay H. Ravaliya2, Elizabeth B. Torres4,5 1Graduate Program in Neuroscience, Rutgers University, 2Department of Biomedical Engineering, Rutgers University, 3Center for Cognitive Science, Rutgers University, 4Department of Psychology, Rutgers University, 5Department of Computer Science, Rutgers University It is unclear how top-down signals from the ventral visual stream affect movement. We developed a paradigm to test motor behavior towards a target on a 3D depth inversion illusion. Significant differences are reported in both deliberate, goal-directed movements and automatic actions under illusory and veridical viewing conditions. Behavior Automated, Quantitative Cognitive/Behavioral Screening of Mice: For Genetics, Pharmacology, Animal Cognition and Undergraduate Instruction C. R. Gallistel1, Fuat Balci1,2, David Freestone1,3, Aaron Kheifets1, Adam King4 1Department of Psychology, Rutgers University, 2Department of Psychology, Koç University, 3Center for Neural Science, New York University, 4Department of Mathematics & Computer Science, Fairfield University Fully automated system for measuring physiologically meaningful properties of the mechanisms mediating spatial localization, temporal localization, duration, rate and probability estimation, risk assessment, impulsivity, and the accuracy and precision of memory, in order to assess the effects of genetic and pharmacological manipulations on foundational mechanisms of cognition in mice. Biology Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins Andrius Masedunskas1,2, Natalie Porat-Shliom1, Muhibullah Tora1, Oleg Milberg1,3, Roberto Weigert1 1Intracellular Membrane Trafficking Unit, Oral and Pharyngeal Cancer Branch National Institute of Dental and Craniofacial Research, National Institutes of Health, 2Department of Biology, University of North Carolina at Chapel Hill, 3Department of Chemical & Biochemical Engineering and Department of Biomedical Engineering, Rutgers University Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice. Biology Analyzing and Building Nucleic Acid Structures with 3DNA Andrew V. Colasanti1, Xiang-Jun Lu2, Wilma K. Olson1 1Department of Chemistry & Chemical Biology and BioMaPS Institute for Quantitative Biology, Rutgers - The State University of New Jersey, 2Department of Biological Sciences, Columbia University The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures. Neuroscience Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents Marie Vandecasteele1,2, S. M.1, Sébastien Royer1,3, Mariano Belluscio1, Antal Berényi1, Kamran Diba1,4, Shigeyoshi Fujisawa1, Andres Grosmark1, Dun Mao1, Kenji Mizuseki1, Jagdish Patel1, Eran Stark1, David Sullivan1, Brendon Watson1, György Buzsáki1 1Center for Molecular and Behavioral Neuroscience, University of New Jersey, 2Center for Interdisciplinary Research in Biology, Collège de France, 3Janelia Farm Research Campus, Howards Hughes Medical Institute, 4Deptartment of Psychology, University of Wisconsin at Milwaukee We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication. Biology In Ovo Electroporation in Embryonic Chick Retina Mohammed M. Islam1, Sung Tae Doh2, Li Cai1,2 1Department of Pharmacology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, 2Department of Biomedical Engineering, Rutgers University The overall goal of this video is to show how to perform targeted retinal injection and in ovo electroporation of DNA/RNA constructs into the chick embryonic retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). This technique is very useful to study gene expression, gene regulation, and morphological change in developing chick retina. Biology Herbivore-induced Blueberry Volatiles and Intra-plant Signaling Cesar R. Rodriguez-Saona1 1Department of Entomology, Rutgers University A push-pull method for collecting plant volatiles is described. The method allows for a comparison of volatiles induced by herbivore feeding, exogenous methyl jasmonate, and mechanical damage. This technique is also used to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants. Biology Isolation and In vitro Activation of Caenorhabditis elegans Sperm Gunasekaran Singaravelu1, Indrani Chatterjee1, Matthew R. Marcello1, Andrew Singson1 1Waksman Institute of Microbiology, Rutgers University A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease. Neuroscience Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales Melinda K. Kutzing*1,2, Christopher G. Langhammer*1,2, Vincent Luo1, Hersh Lakdawala1, Bonnie L. Firestein1 1Department of Cell Biology and Neuroscience, Rutgers University, 2Graduate Program in Biomedical Engineering, Rutgers University We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed. Neuroscience Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila Joanna C. Chiu1,2, Kwang Huei Low1,3, Douglas H. Pike1, Evrim Yildirim1,3, Isaac Edery1,3 1Center for Advanced Biotechnology and Medicine, Rutgers University, 2Current Address: Department of Entomology, College of Agricultural and Environmental Sciences, University of California, Davis, 3Department of Molecular Biology and Biochemistry, Rutgers University We describe procedures for recording daily locomotor activity rhythms of Drosophila and subsequent data analysis. Locomotor activity rhythms are a reliable behavioral output of animal circadian clocks and are used as the standard readout of clock function when studying circadian mutants or examining how the environment regulates the circadian system. Biology Optical Scatter Microscopy Based on Two-Dimensional Gabor Filters Nada N. Boustany1, Robert M. Pasternack1, Jing-Yi Zheng1 1Department of Biomedical Engineering, Rutgers University We demonstrate a dark-field microscopy method based on Gabor-like filtering to measure subcellular dynamics within single living cells. The technique is sensitive to alterations in the structure of organelles, such as mitochondrial fragmentation.