Purdue University View Institution's Website 50 articles published in JoVE Medicine A Reproducible Cartilage Impact Model to Generate Post-Traumatic Osteoarthritis in the Rabbit Julian Dilley*1,2,3, Hessam Noori-Dokht*3,4,6, Abhijit Seetharam1, Margaret Bello1,3, Aaron Nanavaty1,3, Roman M. Natoli2,3, Todd McKinley1,2,3, Zachary Bault5, Diane Wagner2,3,6, Uma Sankar1,3 1Department of Anatomy, Cell Biology and Physiology, Indiana University School of Medicine, 2Department of Orthopaedic Surgery, Indiana University School of Medicine, 3Indiana Center for Musculoskeletal Health, Indiana University School of Medicine, 4School of Mechanical Engineering, Purdue University, 5Large Animal Resource Center, Indiana University School of Medicine, 6Department of Mechanical and Energy Engineering, Indiana University-Purdue University Indianapolis The open medial femoral condyle impact model in rabbits is reliable for studying post-traumatic osteoarthritis (PTOA) and novel therapeutic strategies to mitigate PTOA progression. This protocol generates an isolated cartilage defect of the posterior medial femoral condyle in rabbits using a carriage-based drop tower with an impactor head. Biochemistry Chemical Affinity-Based Isolation of Extracellular Vesicles from Biofluids for Proteomics and Phosphoproteomics Analysis Yi-Kai Liu1, Zhuojun Luo1, Anton Iliuk1,2, Weiguo Andy Tao1,2,3,4 1Department of Biochemistry, Purdue University, 2Tymora Analytical Operations, 3Department of Chemistry, Purdue University, 4Purdue Institute for Cancer Research, Purdue University The present protocol provides detailed descriptions for the efficient isolation of urinary extracellular vesicles utilizing functionalized magnetic beads. Moreover, it encompasses subsequent analyses, including western blotting, proteomics, and phosphoproteomics. Biology Tracking miRNA Release into Extracellular Vesicles using Flow Cytometry Humna Hasan1, Andrea L. Kasinski1,2 1Department of Biological Sciences, Purdue University, 2Purdue Institute for Cancer Research, Purdue University Here, we describe a straightforward protocol that enables in vitro assessment of the abundance of fluorescently labeled microRNAs to study the dynamics of microRNA packaging and export into extracellular vesicles (EVs). Biology Agrobacterium tumefaciens-Mediated Genetic Transformation of Narrowleaf Plantain Hannah Levengood1, Yanxia Dou1, Jinping Fan1, Anna Bajszar2, Jing Huang1, Syed Mohsin Abbas3, Yun Zhou4, Cankui Zhang1 1Department of Agronomy, Center for Plant Biology, Purdue University, 2Department of Biology, Purdue University, 3Department of Horticulture, Faculty of Agricultural Sciences, University of the Punjab, 4Department of Botany and Plant Pathology, Center for Plant Biology, Purdue University Because of its versatile application as a model species in various fields of study, there is a need for a genetic transformation toolkit in narrowleaf plantain (Plantago lanceolata). Here, using Agrobacterium tumefaciens-mediated transformation, a protocol is presented that results in stable transgenic lines with a transformation efficiency of 20%. Chemistry Direct Comparison of Hyperspectral Stimulated Raman Scattering and Coherent Anti-Stokes Raman Scattering Microscopy for Chemical Imaging Matthew G. Clark1, Kent A. Brasseale III1, Gil A. Gonzalez1, Gregory Eakins1, Chi Zhang1 1Department of Chemistry, Purdue University This paper directly compares the resolution, sensitivity, and imaging contrasts of stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS) integrated into the same microscope platform. The results show that CARS has a better spatial resolution, SRS gives better contrasts and spectral resolution, and both methods have similar sensitivity. Neuroscience ROS Live Cell Imaging During Neuronal Development Aslihan Terzi1,2, S. M. Sabbir Alam1,2, Daniel M. Suter1,2,3 1Department of Biological Sciences, Purdue University, 2Purdue Institute for Integrative Neuroscience, Purdue University, 3Bindley Bioscience Center, Purdue University This protocol describes the use of a genetically encoded hydrogen peroxide (H2O2)-biosensor in cultured zebrafish neurons and larvae for assessing the physiological signaling roles of H2O2 during nervous system development. It can be applied to different cell types and modified with experimental treatments to study reactive oxygen species (ROS) in general development. Medicine Use of an Integrated Low-Flow Anesthetic Vaporizer, Ventilator, and Physiological Monitoring System for Rodents Krista Bigiarelli1, Luke E. Schepers2, Arvin H. Soepriatna2, Dave FitzMiller3, Craig J. Goergen2 1Pre-Clinical Research and Development, Kent Scientific Corporation, 2Weldon School of Biomedical Engineering, Purdue University, 3Marketing Research and Development, Kent Scientific Corporation Here, we present a protocol to safely and effectively administer anesthetic gas to mice using a digital, low flow anesthesia system with integrated ventilator and physiological monitoring modules. Biochemistry Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis Chuan-Chih Hsu1,3, Chia-Feng Tsai2, W. Andy Tao3,4, Pengcheng Wang5 1Department of Plant Biology, Carnegie Institute for Science, 2Graduate School of Pharmaceutical Sciences, Kyoto University, 3Department of Biochemistry, Purdue University, 4Department of Chemistry, Purdue University, 5Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences Presented here is a phosphoproteomic approach, namely stop and go extraction tip based phosphoproteomic, which provides high-throughput and deep coverage of Arabidopsis phosphoproteome. This approach delineates the overview of osmotic stress signaling in Arabidopsis. Medicine Leveraging Turbidity and Thromboelastography for Complementary Clot Characterization Ziqian Zeng1,2, Tanmaye Nallan Chakravarthula1,2, Nathan J. Alves1,2 1Emergency Medicine Department, Indiana University School of Medicine, 2Weldon School of Biomedical Engineering, Purdue University Fibrin is responsible for clot formation during hemostasis and thrombosis. Turbidity assays and thromboelastograhy (TEG) can be utilized as synergistic tools that provide complementary assessment of a clot. These two techniques together can give more insight into how clotting conditions affect fibrin clot formation. Biology The CryoAPEX Method for Electron Microscopy Analysis of Membrane Protein Localization Within Ultrastructurally-Preserved Cells Elaine M. Mihelc1, Stephanie Angel2, Robert V. Stahelin2,3,4, Seema Mattoo1,4,5,6 1Department of Biological Sciences, Purdue University, 2Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, 3Purdue Institute of Inflammation, Immunology and Infectious Disease, Purdue University, 4Bindley Biosciences Center, Purdue University, 5Purdue Institute of Integrative Neuroscience, Purdue University, 6Purdue Center for Cancer Research, Purdue University This protocol describes the cryoAPEX method, in which an APEX2-tagged membrane protein can be localized by transmission electron microscopy within optimally-preserved cell ultrastructure. Engineering Accurate Determination of the Equilibrium Surface Tension Values with Area Perturbation Tests Jaeyub Chung1, Bryan W. Boudouris1,2, Elias I. Franses1 1Charles D. Davidson School of Chemical Engineering, Purdue University, 2Department of Chemistry, Purdue University Two protocols for determining the equilibrium surface tension (EST) values using the emerging bubble method (EBM) and the spinning bubble method (SBM) are presented for a surfactant-containing aqueous phase against air. Chemistry Multiscale Sampling of a Heterogeneous Water/Metal Catalyst Interface using Density Functional Theory and Force-Field Molecular Dynamics Cameron J. Bodenschatz*1, Xiaohong Zhang*1, Tianjun Xie*1, Jeremy Arvay1,2, Sapna Sarupria1, Rachel B. Getman1 1Department of Chemical and Biomolecular Engineering, Clemson University, 2Davidson School of Chemical Engineering, Purdue University The goal of the protocol presented here is to generate and sample trajectories of configurations of liquid water molecules around catalytic species on a flat transition metal surface. The sampled configurations can be used as starting structures in quantum mechanics-based methods. Developmental Biology Confocal Live Imaging of Shoot Apical Meristems from Different Plant Species Yuan Geng1,2, Yun Zhou1,2 1Department of Botany and Plant Pathology, Purdue University, 2Purdue Center for Plant Biology, Purdue University This protocol presents how to live image and analyze the shoot apical meristems from different plant species using laser scanning confocal microscopy. Biochemistry Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale Melaku Garsamo1,3, Yun Zhou2,3, Xing Liu1,3 1Department of Biochemistry, Purdue University, 2Department of Botany and Plant Pathology, Purdue University, 3Center for Plant Biology, Purdue University Protein-protein interactions are critical for biological systems, and studies of the binding kinetics provide insights into the dynamics and function of protein complexes. We describe a method that quantifies the kinetic parameters of a protein complex using fluorescence resonance energy transfer and the stopped-flow technique. Biology Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes Carlos A Brito-Sierra*1, Jasleen Kaur*1, Catherine A. Hill1,2 1Department of Entomology, Purdue University, 2Purdue Institute for Inflammation, Immunology and Infectious Disease, Purdue University Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion. Behavior Using Rapid Serial Visual Presentation to Measure Set-Specific Capture, a Consequence of Distraction While Multitasking Katherine S. Moore1, Elizabeth A. Wiemers2, Ariel Kershner1, Korissa Belville1, Jaimie Jasina1, Aziza Ransome1, Jessica Avanzato1 1Department of Psychology, Arcadia University, 2Department of Psychological Sciences, Purdue University This method uses a dynamic visual display to index costs of distraction during visual search, including both "contingent attentional capture" and "set-specific capture," which is a cost of distraction that occurs when the participants maintain multiple search goals simultaneously. This method has revealed basic mechanisms and limitations of visual attention. Genetics Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio Erin L. Sorlien1, Mary A. Witucki1, Joseph Ogas1 1Department of Biochemistry, Purdue University Targeted genome editing in the model system Danio rerio (zebrafish) has been greatly facilitated by the emergence of CRISPR-based approaches. Herein, we describe a streamlined, robust protocol for generation and identification of CRISPR-derived nonsense alleles that incorporates the heteroduplex mobility assay and identification of mutations using next-generation sequencing. Engineering Three-electrode Coin Cell Preparation and Electrodeposition Analytics for Lithium-ion Batteries Robert D. Minter*1, Daniel Juarez-Robles*2, Conner Fear2, Yevgen Barsukov3, Partha P. Mukherjee2 1Department of Mechanical Engineering, Texas A&M University, 2School of Mechanical Engineering, Purdue University, 3Battery Management Systems, Texas Instruments Inc. Three-electrode cells are useful in studying the electrochemistry of lithium-ion batteries. Such an electrochemical setup allows the phenomena associated with the cathode and anode to be decoupled and examined independently. Here, we present a guide for construction and use of a three-electrode coin cell with emphasis on lithium plating analytics. Behavior Tickling, a Technique for Inducing Positive Affect When Handling Rats Sylvie Cloutier1,2, Megan R. LaFollette3, Brianna N. Gaskill3, Jaak Panksepp1, Ruth C. Newberry4 1Center for the Study of Animal Well-being, Department of Integrative Physiology and Neuroscience, Washington State University, 2Canadian Council on Animal Care, 3Department of Animal Sciences, Center for Animal Welfare Science, College of Agriculture, Purdue University, 4Department of Animal and Aquacultural Sciences, Faculty of Biosciences, Norwegian University of Life Sciences This article demonstrates the standardized application of playful handling, a tickling technique designed to mimic rat rough-and-tumble play. This technique is effective at reducing fearful reactions to humans and generating positive affect when rats are handled for common husbandry activities and medical and research procedures such as injection. Developmental Biology Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors Martin R. Silic1, GuangJun Zhang1,2 1Department of Comparative Pathobiology, Purdue University, 2Purdue University Center for Cancer Research, Purdue Institute for Inflammation, Immunology and Infectious Diseases (PI4D), Purdue Institute for Integrative Neuroscience (PIIN), Purdue University Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo. Medicine Differential Effects of Lipid-lowering Drugs in Modulating Morphology of Cholesterol Particles Shanmugavel Madasamy1, David Liu1, Jason Lundry1, Benjamin Alderete2, Raymond Kong2, J. Paul Robinson3, Alan H.B. Wu1,4, Edward P. Amento5 1Plaxgen Inc, 2Millipore Sigma, 3Cytometry Laboratories, Purdue University, 4San Francisco General Hospital, 2M16 Clinical Chemistry, University of California, San Francisco, 5Molecular Medicine Research Institute The objective of this study was to evaluate in vitro lipid-lowering drug effects in modulating the morphology of cholesterol particles. Comparison of lipid-lowering drugs revealed variations in their effect in modulating the morphological features of cholesterol particles. Genetics Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes Elizabeth G. Porter1, Katelyn E. Connelly1, Emily C. Dykhuizen1 1Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University Sequential salt extraction of chromatin bound proteins is a useful tool for determining the binding properties of large protein complexes. This method can be employed to evaluate the role of individual subunits or domains in the overall affinity of a protein complex to bulk chromatin. Neuroscience Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices Qiuyu Wu1,2, Alexander A. Chubykin1,2 1Department of Biological Sciences, Purdue University, 2Purdue Institute for Integrative Neuroscience, Purdue University This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment. Chemistry Synthesis and Testing of Supported Pt-Cu Solid Solution Nanoparticle Catalysts for Propane Dehydrogenation Zixue Ma1, Zhenwei Wu1, Jeffrey T. Miller1 1Davidson School of Chemical Engineering, Purdue University A convenient method for the synthesis of 2 nm supported bimetallic nanoparticle Pt-Cu catalysts for propane dehydrogenation is reported here. In situ synchrotron X-ray techniques allow for the determination of the catalyst structure, which is typically unobtainable using laboratory instruments. Medicine Comparing the Effects of Electronic Cigarette Vapor and Cigarette Smoke in a Novel In Vivo Exposure System Anthony N. Hage1, Will Krause1, Angela Mathues1, Luke Krasner2, Seth Kasten1, Jonathan L. Eliason1,3, Abhijit Ghosh1 1Jobst Vascular Research Laboratory, University of Michigan Medical School, 2Department of Engineering, Purdue University, 3Department of Surgery, Section of Vascular Surgery, University of Michigan Health System This protocol describes a method for exposing rodents to electronic cigarette vapor (E-vapor) and cigarette smoke. Exposure chambers are constructed by modifying anesthesia chambers with an automated pumping system that delivers E-vapor or cigarette smoke to rodents. This system can easily be modified to accommodate many experimental endpoints. Bioengineering Quantification of Strain in a Porcine Model of Skin Expansion Using Multi-View Stereo and Isogeometric Kinematics Adrian Buganza Tepole1, Elbert E. Vaca2, Chad A. Purnell2, Michael Gart2, Jennifer McGrath2, Ellen Kuhl3, Arun K. Gosain2 1Mechanical Engineering, Purdue University, 2Division of Plastic Surgery, Ann and Robert H. Lurie Children's Hospital of Chicago, Northwestern University Feinberg School of Medicine, 3Mechanical Engineering, Bioengineering, Cardiothoracic Surgery, Stanford University This protocol uses multi-view stereo to generate three-dimensional (3D) models out of uncalibrated sequences of photographs, making it affordable and adjustable to a surgical setting. Strain maps between the 3D models are quantified with spline-based isogeometric kinematics, which facilitate representation of smooth surfaces over coarse meshes sharing the same parameterization. Bioengineering Dissection, MicroCT Scanning and Morphometric Analyses of the Baculum Nicholas G. Schultz1, Erik Otárola-Castillo2, Matthew D. Dean1 1Molecular and Computational Biology, University of Southern California (USC), 2Purdue University Many biological structures lack easily definable landmarks, making it difficult to apply modern morphometric methods. Here we illustrate methods to study the mouse baculum (a bone in the penis), including dissection and microCT scanning, followed by computational methods to define semi-landmarks that are used to quantify size and shape variation. Medicine Use of a Low-flow Digital Anesthesia System for Mice and Rats Amelia R. Adelsperger1, Krista J. Bigiarelli-Nogas2, Irina Toore2, Craig J. Goergen1 1Weldon School of Biomedical Engineering, Purdue University, 2Pre-Clinical Research and Development, Kent Scientific Corporation Here, we present a protocol to more safely and efficiently administer anesthetic gas to mice using a digital, low flow anesthesia system utilizing a syringe-driven direct injection vaporizer. Chemistry From Constructs to Crystals – Towards Structure Determination of β-barrel Outer Membrane Proteins Nicholas Noinaj1, Stephen Mayclin2, Ann M. Stanley2,3, Christine C. Jao2,3, Susan K. Buchanan2 1Department of Biological Sciences, Markey Center for Structural Biology, Purdue University, 2National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health, 3National Institute of General Medical Sciences (NIGMS), National Institutes of Health β-barrel outer membrane proteins (OMPs) serve many functions within the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Here, we hope to alleviate a known bottleneck in structural studies by presenting protocols for the production of β-barrel OMPs in sufficient quantities for structure determination by X-ray crystallography or NMR spectroscopy. Engineering Data Acquisition Protocol for Determining Embedded Sensitivity Functions Janette J. Meyer1, Douglas E. Adams1, Janene Silvers2 1Laboratory for Systems Integrity and Reliability (LASIR), Vanderbilt University, 2Mechanical Engineering, Purdue University The data acquisition procedure for determining embedded sensitivity functions is described. Data is acquired and representative results are shown for a residential scale wind turbine blade. Chemistry Mass Spectrometric Approaches to Study Protein Structure and Interactions in Lyophilized Powders Balakrishnan S. Moorthy*1, Lavanya K. Iyer*1, Elizabeth M. Topp1 1Department of Industrial and Physical Pharmacy, Purdue University Here, we present detailed protocols for solid-state amide hydrogen/deuterium exchange mass spectrometry (ssHDX-MS) and solid-state photolytic labeling mass spectrometry (ssPL-MS) for proteins in solid powders. The methods provide high-resolution information on protein conformation and interactions in the amorphous solid-state, which may be useful in formulation design. Biology Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants Xiaohong Zhu1, Aaron Taylor2, Shenyu Zhang1, Dayong Zhang1,3, Ying Feng1,4, Gaimei Liang1,5, Jian-Kang Zhu1,6 1Department of Horticulture and Landscape Architecture, Purdue University, 2Bindley Bioscience Center, Purdue University, 3Institute of Biotechnology, Jiangsu Academy of Agricultural Sciences, 4College of Environmental & Resource Science, Zhejiang University, 5Dryland Agriculture Research Centre, Shanxi Academy of Agricultural Sciences, 6Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12. Engineering Real-Time DC-dynamic Biasing Method for Switching Time Improvement in Severely Underdamped Fringing-field Electrostatic MEMS Actuators Joshua Small1, Adam Fruehling2, Anurag Garg3, Xiaoguang Liu1, Dimitrios Peroulis3 1Department of Electrical and Computer Engineering, University of California, Davis, 2Digital Light Projection (DLP) Technology Development, Texas Instruments, 3Birck Nanotechnology Center and the Department of Electrical and Computer Engineering, Purdue University The robust device design of fringing-field electrostatic MEMS actuators results in inherently low squeeze-film damping conditions and long settling times when performing switching operations using conventional step biasing. Real-time switching time improvement with DC-dynamic waveforms reduces the settling time of fringing-field MEMS actuators when transitioning between up-to-down and down-to-up states. Biology Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis Jingqun Ma1, Vikki Marie Weake1 1Department of Biochemistry, Purdue University Drosophila tissues often contain a heterogeneous mixture of cell types. To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation. Medicine A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies Mukti R. Parikh1, Andrew R. Belch2,3, Linda M Pilarski2,3, Julia Kirshner1 1Department of Biological Sciences, Purdue University, 2Department of Oncology, University of Alberta, 3Cross Cancer Institute In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue. Bioengineering Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications Jason M. Conley1, Tarsis F. Brust1, Ruqiang Xu1, Kevin D. Burris2, Val J. Watts1 1Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, 2Quantitative Biology, Eli Lilly and Company Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening. Environment Helminth Collection and Identification from Wildlife Maria S Sepulveda1, John M Kinsella2 1Department of Forestry and Natural Resources, Purdue University, 2Helm West Laboratory Wild animals are commonly parasitized by a wide range of helminths. The four major types of helminths are “roundworms” (nematodes), “thorny-headed worms” (acanthocephalans), “flukes” (trematodes), and “tapeworms” (cestodes). Here we describe how helminths are collected from a vertebrate animal and how they are preserved and taxonomically identified. Bioengineering Multi-analyte Biochip (MAB) Based on All-solid-state Ion-selective Electrodes (ASSISE) for Physiological Research Wan W. Amani Wan Salim1, Michael A. Zeitchek1, Andrew C. Hermann1, Antonio J. Ricco2, Ming Tan2, Florian Selch2, Erich Fleming2, Brad M. Bebout2, Mamoun M. Bader3, Aeraj ul Haque4, D. Marshall Porterfield5 1Department of Agricultural and Biological Engineering, Birck-Bindley Physiological Sensing Facility, Purdue University, 2NASA Ames Research Center, 3Department of Chemistry, Pennsylvania State University Hazleton, 4Cooley LLP, 5NASA Life and Physical Sciences, Human Exploration and Operations Mission Directorate, NASA Headquarters All-solid-state ion-selective electrodes (ASSISEs) constructed from a conductive polymer (CP) transducer provide several months of functional lifetime in liquid media. Here, we describe the fabrication and calibration process of ASSISEs in a lab-on-a-chip format. The ASSISE is demonstrated to have maintained a near-Nernstian slope profile after prolonged storage in complex biological media. Neuroscience Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue Andrew J. Woolley1, Himanshi A. Desai1, Janak Gaire2, Andrew L. Ready1, Kevin J. Otto1,2 1Weldon School of Biomedical Engineering, Purdue University, 2Department of Biological Sciences, Purdue University Here we present a histological method for capturing, labeling, optically clearing, and imaging the intact brain tissue interface around chronically implanted microdevices in rodent brain tissue. Results from the techniques comprising this method are useful for understanding the impact of various penetrating brain-implants on their surrounding tissue. Neuroscience Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices Staci E. Engle1, Hilary J. Broderick1, Ryan M. Drenan1 1Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University In this paper, we describe a useful method to study ligand-gated ion channel function in neurons of acutely isolated brain slices. This method involves the use of a drug-filled micropipette for local application of drugs to neurons recorded using standard patch clamp techniques. Engineering Investigation of Early Plasma Evolution Induced by Ultrashort Laser Pulses Wenqian Hu1, Yung C. Shin1, Galen B. King1 1Mechanical Engineering, Purdue University An experimental method to examine the early plasma evolution induced by ultrashort laser pulses is described. Using this method, high quality images of early plasma are obtained with high temporal and spatial resolutions. A novel integrated atomistic model is used to simulate and explain the mechanisms of early plasma. Neuroscience Voltage Biasing, Cyclic Voltammetry, & Electrical Impedance Spectroscopy for Neural Interfaces Seth J. Wilks1, Tom J. Richner2, Sarah K. Brodnick2, Daryl R. Kipke3, Justin C. Williams2, Kevin J. Otto1,4 1Weldon School of Biomedical Engineering, Purdue University, 2Biomedical Engineering, University of Wisconsin-Madison, 3Biomedical Engineering, University of Michigan, 4Department of Biological Sciences, Purdue University The electrode-tissue interface of neural recording electrodes can be characterized with electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Application of voltage biasing changes the electrochemical properties of the electrode-tissue interface and can improve recording capability. Voltage biasing, EIS, CV, and neural recordings are complementary. Neuroscience Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays Kristen N. Fantetti1, Donna M. Fekete1 1Department of Biological Sciences, Purdue University We demonstrate how to dissect and culture chick E4 statoacoustic ganglion and E6 spinal cord explants. Explants are cultured under serum-free conditions in 3D collagen gels for 24 hours. Neurite responsiveness is tested with growth factor-supplemented medium and with protein-coated beads. Immunology and Infection Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells Sophie Moreau-Marquis1, Carly V. Redelman2, Bruce A. Stanton1, Gregory G. Anderson2 1Department of Physiology, Dartmouth College, 2Department of Biology, Indiana University Purdue University Indianapolis This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity. Biology Microdissection of Zebrafish Embryonic Eye Tissues Liyun Zhang1, Yuk Fai Leung1 1Department of Biological Sciences, Purdue University This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos. Biology Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast Debarati Mukherjee1, Arpita Sen1, R. Claudio Aguilar1 1Department of Biological Sciences and Purdue Center for Cancer Research, Purdue University Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Biology A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples Eric Johansen1, Birgit Schilling2, Michael Lerch1, Richard K. Niles1, Haichuan Liu1, Bensheng Li2, Simon Allen1, Steven C. Hall1, H. Ewa Witkowska1, Fred E. Regnier3, Bradford W. Gibson2, Susan J. Fisher1, Penelope M. Drake1 1Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco - UCSF, 2Buck Institute for Age Research, 3Department of Chemistry, Purdue University Lectin-conjugated POROS beads were employed for HPLC. Glycopeptide standards served as positive and negative controls. MARS-14 depleted, trypsin-digested human plasma was chromatographed and flow-through (FT) and bound fractions collected for ESI-LC-MS/MS analyses. Glycopeptides were enriched in the bound fraction as compared to FT. Biology Global Gene Expression Analysis Using a Zebrafish Oligonucleotide Microarray Platform Samuel M. Peterson1, Jennifer L. Freeman1 1School of Health Sciences, Purdue University Gene microarrays are powerful tools in gene expression profiling at a genome-wide level. This technology has application in a variety of biological disciplines including developmental biology and toxicology. In this video, we detail a protocol for global gene expression analysis using a comprehensive oligonucleotide microarray platform for the zebrafish. Biology RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis Samuel M. Peterson1, Jennifer L. Freeman1 1School of Health Sciences, Purdue University The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis. Biology Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones Aih Cheun Lee1, Boris Decourt1, Daniel M. Suter1 1Department of Biological Sciences, Purdue University Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.
