Arizona State University View Institution's Website 34 articles published in JoVE Engineering Metal-Assisted Electrochemical Nanoimprinting of Porous and Solid Silicon Wafers Aliaksandr Sharstniou1, Stanislau Niauzorau1, Ashlesha Junghare1, Bruno P. Azeredo1 1The Polytechnic School, Arizona State University A protocol for metal-assisted chemical imprinting of 3D microscale features with sub-20 nm shape accuracy into solid and porous silicon wafers is presented. Biology Topical Application Bioassay to Quantify Insecticide Toxicity for Mosquitoes and Fruit Flies Brook M. Jensen1, Rachel A. Althoff1, Sarah E. Rydberg1, Emma N. Royster1, Alden Estep2, Silvie Huijben1 1Center for Evolution and Medicine, School of Life Sciences, Arizona State University, 2United States Department of Agriculture, Agricultural Research Service, Center for Medical, Agricultural and Veterinary Entomology We describe the methodology and importance of the topical application bioassay to measure insecticide susceptibility in mosquitoes and fruit flies. The presented assay is high-throughput, utilizes insect mass-thus allowing for calculating a mass-relativized lethal dose instead of concentration-and likely has lower variability than other similar methods. Genetics Optimized Bone Sampling Protocols for the Retrieval of Ancient DNA from Archaeological Remains Cody E. Parker1,2, Kirsten I. Bos1,3, Wolfgang Haak1,3, Johannes Krause1,3 1Department of Archaeogenetics, Max Planck Institute for the Science of Human History, 2School of Human Evolution and Social Change, Arizona State University, 3Department of Archaeogenetics, Max Planck Institute for Evolutionary Anthropology The protocol presents a series of best practice protocols for the collection of bone powder from eight recommended anatomical sampling locations (specific locations on a given skeletal element) across five different skeletal elements from medieval individuals (radiocarbon dated to a period of ca. 1040-1400 CE, calibrated 2-sigma range). Biochemistry Sample Preparation using a Lipid Monolayer Method for Electron Crystallographic Studies Chloe D. Truong1,2, Dewight R. Williams3, Mary Zhu1, Joseph Che-Yen Wang4, Po-Lin Chiu1,2 1School of Molecular Sciences, Arizona State University, 2Center for Applied Structural Discovery, Biodesign Institute, Arizona State University, 3Eyring Materials Center, Arizona State University, 4Department of Microbiology and Immunology, The Pennsylvania State University Lipid monolayers have been used as a foundation for forming two-dimensional (2D) protein crystals for structural studies for decades. They are stable at the air-water interface and can serve as a thin supporting material for electron imaging. Here we present the proven steps on preparing lipid monolayers for biological studies. Bioengineering Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform Jaimeson Veldhuizen1, Mehdi Nikkhah1,2 1School of Biological and Health Systems Engineering, Arizona State University, 2Biodesign Virginia G. Piper Center for Personalized Diagnostics, Arizona State University The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling. Biology Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana Aigerim S. Kamzina*1,2,3, Michelle P. DiPalma*1,2,3, Joseph G. L. Hunter1,2,3, Andrew G. Diamos1,2,4, Boyd Armer2,3, Tsafrir S. Mor1,2,3, Hugh S. Mason1,2,3 1Center for Immunotherapy, Vaccines and Virotherapy, The Biodesign Institute, Arizona State University, 2School of Life Sciences, Arizona State University, 3Molecular Biosciences/Biotechnology Undergraduate Program, Arizona State University, 4Department of Microbiology and Immunology, University of Michigan Medical School We describe here a simple method for expression, extraction, and purification of recombinant human IgG fused to GFP in Nicotiana benthamiana. This protocol can be extended to purification and visualization of numerous proteins that utilize column chromatography. Moreover, the protocol is adaptable to the in-person and virtual college teaching laboratory, providing project-based exploration. Chemistry Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay Jesús Aguilar Diaz De Leon1, Chad R. Borges 1School of Molecular Sciences, The Biodesign Institute - Center for Personalized Diagnostics, Arizona State University The goal of the thiobarbituric acid reactive substances assay is to assess oxidative stress in biological samples by measuring the production of lipid peroxidation products, primarily malondialdehyde, using visible wavelength spectrophotometry at 532 nm. The method described here can be applied to human serum, cell lysates, and low density lipoproteins. Neuroscience Anti-RDL and Anti-mGlutR1 Receptors Antibody Testing in Honeybee Brain Sections using CRISPR-Cas9 Irina Sinakevitch1,2, Zev Kurtzman*2, Hyun G. Choi*2, David Arturo Ruiz Pardo3, Romain A. Dahan2, Nathaniel Klein1, Branimir Bugarija4, Erik Wendlandt4, Brian H. Smith2 1Department of Neuroscience, University of Arizona, 2School of Life Sciences, Arizona State University, 3Department of Scientific and Technologic Investigations, University of Sonora, 4Integrated DNA Technologies, Inc. Presented here is a protocol to use the CRISPR-Cas9 system for reducing the production of a protein in the adult honeybee brain to test antibody specificity. Bioengineering Ovarian Cancer Detection Using Photoacoustic Flow Cytometry Joel F. Lusk1, Christopher Miranda1, Barbara S. Smith1 1School of Biological and Health Systems Engineering, Arizona State University A protocol is presented to detect circulating ovarian tumor cells utilizing a custom-made photoacoustic flow system and targeted folic acid-capped copper sulfide nanoparticles. Biochemistry Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays Fernanda Festa1,2, Joshua Labaer1 1Virginia G. Piper Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, 2Department of Pediatrics, and Biochemistry/Molecular Biology, College of Medicine, Pennsylvania State University A detailed protocol for the generation of self-assembled human protein microarrays for the screening of kinase inhibitors is presented. Engineering X-ray Beam Induced Current Measurements for Multi-Modal X-ray Microscopy of Solar Cells Christina Ossig1,3, Tara Nietzold2, Bradley West2, Mariana Bertoni2, Gerald Falkenberg1, Christian G. Schroer1,3, Michael E. Stuckelberger1 1Deutsches Elektronen-Synchrotron, 2School of Electrical, Computer and Energy Engineering, Arizona State University, 3Department Physik, Universität Hamburg A setup for X-ray beam induced current measurements at synchrotron beamlines is described. It unveils the nanoscale performance of solar cells and extends the suite of techniques for multi-modal X-ray microscopy. From wiring to signal-to-noise optimization, it is shown how to perform state-of-the-art XBIC measurements at a hard X-ray microprobe. Bioengineering Using Nanoplasmon-Enhanced Scattering and Low-Magnification Microscope Imaging to Quantify Tumor-Derived Exosomes Meihua Wan1,2, Pouya Amrollahi2,3, Dali Sun4, Christopher Lyon2,3, Tony Y. Hu2,3 1Department of Integrated Traditional Chinese and Western Medicine, West China Hospital of Sichuan University, 2Virginia G. Piper Biodesign Center for Personalized Diagnostics, The Biodesign Institute, Arizona State University, 3School of Biological and Health Systems Engineering, Arizona State University, 4Department of Electrical and Computer Engineering, North Dakota State University Clinical translation of exosome-derived biomarkers for diseased and malignant cells is hindered by the lack of rapid and accurate quantification methods. This report describes the use of low-magnification dark-field microscope images to quantify specific exosome subtypes in small volume serum or plasma samples. Environment A Precise and Autonomous System for the Detection of Insect Emergence Patterns Meghan M. Bennett1,2, Joseph P. Rinehart3, George D. Yocum3, Ian Yocum3 1Department of Biological Sciences, North Dakota State University, 2School of Life Sciences, Arizona State University, 3Red River Valley Agricultural Research Center, USDA-ARS Measurement of insect emergence patterns requires precision. Existing systems are only semi-automated and sample size is limited. We addressed these issues by designing a system using microcontrollers to precisely measure the time of emergence of large numbers of emerging insects. Engineering Developing High Performance GaP/Si Heterojunction Solar Cells Chaomin Zhang1, Ehsan Vadiee1, Som Dahal1, Richard R. King1, Christiana B. Honsberg1 1School of Electrical, Computer, and Energy Engineering, Arizona State University Here, we present a protocol to develop high-performance GaP/Si heterojunction solar cells with a high Si minority-carrier lifetime. Genetics Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms Clark Fritsch1,2, Jean-Francois Pierre Gout3,4, Marc Vermulst1 1 This protocol provides researchers with a new tool to monitor the fidelity of transcription in multiple model organisms. Immunology and Infection Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion James J. Faust1,2, Wayne Christenson3,4,5, Kyle Doudrick6, John Heddleston7, Teng-Leong Chew7, Marko Lampe8, Arnat Balabiyev1,2, Robert Ros3,4,5, Tatiana P. Ugarova1,2 1Center for Metabolic and Vascular Biology, Mayo Clinic, 2Molecular and Cellular Biosciences, School of Life Sciences, Arizona State University, 3Department of Physics, Arizona State University, 4Center for Biological Physics, Arizona State University, 5Biodesign Institute, Arizona State University, 6Department of Civil and Environmental Engineering and Earth Sciences, University of Notre Dame, 7Advanced Imaging Center, HHMI Janelia Research Campus, 8Advanced Light Microscopy Facility, European Molecular Biology Laboratory This protocol describes the fabrication of optical-quality glass surfaces adsorbed with compounds containing long-chain hydrocarbons that can be used to monitor macrophage fusion of living specimens and enables super-resolution microscopy of fixed specimens. Chemistry Quantifying X-Ray Fluorescence Data Using MAPS Tara Nietzold1, Bradley M. West2, Michael Stuckelberger2, Barry Lai3, Stefan Vogt3, Mariana I Bertoni2 1School for Engineering of Matter, Transport, and Energy, Arizona State University, 2School of Electrical, Computer, and Energy Engineering, Arizona State University, 3Advanced Photon Source, Argonne National Laboratory Here, we demonstrate the use of the X-ray fluorescence fitting software, MAPS, created by Argonne National Laboratory for the quantification of fluorescence microscopy data. The quantified data that results is useful for understanding the elemental distribution and stoichiometric ratios within a sample of interest. Neuroscience SwarmSight: Real-time Tracking of Insect Antenna Movements and Proboscis Extension Reflex Using a Common Preparation and Conventional Hardware Justas Birgiolas1, Christopher M. Jernigan1, Richard C. Gerkin1, Brian H. Smith1, Sharon M. Crook1,2 1School of Life Sciences, Arizona State University, 2School of Mathematical and Statistical Sciences, Arizona State University This protocol describes steps for using the novel software, SwarmSight, for frame-by-frame tracking of insect antenna and proboscis positions from conventional web camera videos using conventional computers. The free, open-source software processes frames about 120 times faster than humans and performs at better than human accuracy. Behavior Assessing Working Memory in Children: The Comprehensive Assessment Battery for Children – Working Memory (CABC-WM) Kathryn Cabbage1, Shara Brinkley2, Shelley Gray2, Mary Alt3, Nelson Cowan4, Samuel Green5, Trudy Kuo6, Tiffany P. Hogan1 1Communication Sciences and Disorders, MGH Institute of Health Professions, 2Speech and Hearing Science, Arizona State University, 3Speech, Language, and Hearing Sciences, University of Arizona, 4Department of Psychological Sciences, University of Missouri-Columbia, 5Sanford School of Social and Family Dynamics, Arizona State University, 6School of Social and Behavioral Sciences, New College of Interdisciplinary Arts and Sciences, Arizona State University - West Working memory predicts a significant amount of variance for a variety of cognitive tasks, including speaking, reading, and writing. However, few tools are available to assess working memory in children. We present an innovative, computer-based battery that comprehensively assesses different components of working memory in school-age children. Biochemistry Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography Andrii Ishchenko1,2, Vadim Cherezov1,2, Wei Liu3 1The Bridge Institute, University of Southern California, 2Department of Chemistry, University of Southern California, 3School of Molecular Sciences, Center for Applied Structural Discovery at the Biodesign Institute, Arizona State University We describe procedures for the preparation and delivery of membrane protein microcrystals in lipidic cubic phase for serial crystallography at X-ray free-electron lasers and synchrotron sources. These protocols can also be applied for incorporation and delivery of soluble protein microcrystals, leading to substantially reduced sample consumption compared to liquid injection. Chemistry Glycan Node Analysis: A Bottom-up Approach to Glycomics Sahba Zaare1, Jesús S. Aguilar1, Yueming Hu1, Shadi Ferdosi1, Chad R. Borges1 1Department of Chemistry & Biochemistry, The Biodesign Institute – Center for Personalized Diagnostics, Arizona State University This article presents an enhanced form of a novel bottom-up glycomics technique designed to analyze the pooled compositional profile of glycans in unfractionated biofluids through the chemical breakdown of glycans into their constituent linkage-specific monosaccharides for detection by GC-MS. Potential applications include early detection of cancer and other glycan-affective disorders. Biology Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay Justin M. Wolter1,2, Kasuen Kotagama1,2, Cody S. Babb2, Marco Mangone1,2 1School of Life Sciences, Arizona State University, 2Biodesign Institute, Arizona State University Luminescent identification of functional elements in 3’ untranslated regions (3’UTRs) (3’LIFE) is a technique to identify functional regulation in 3’UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression. Neuroscience Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons Megan N. Evilsizor1,2, Helen F. Ray-Jones1,3, Jonathan Lifshitz1,2,4,5, Jenna Ziebell1,2 1Department of Child Health, University of Arizona College of Medicine - Phoenix, 2 This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brains, using markers for microglia and neuronal elements as an example. Bioengineering Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis Stephanie M. Williams1, Lalitha Venkataraman1, Huilai Tian1, Galam Khan2, Brent T. Harris2,3, Michael R. Sierks1 1School for Engineering of Matter, Transport and Energy, Arizona State University, 2Department of Neurology, Georgetown University Medical Center, 3Department of Pathology, Georgetown University Medical Center Using atomic force microscopy in combination with biopanning technology we created a negative and positive biopanning system to acquire antibodies against disease-specific protein variants present in any biological material, even at low concentrations. We were successful in obtaining antibodies to TDP-43 protein variants involved in Amyotrophic Lateral Sclerosis. Biology Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment (Kartchner Caverns, AZ, USA) Lin Zhang1, Katleen Vranckx2, Koen Janssens2, Todd R. Sandrin1 1School of Mathematical and Natural Sciences, Arizona State University, 2Applied Maths NV This work details procedures for rapid identification of bacteria using MALDI-TOF MS. The identification procedures include spectrum acquisition, database construction, and follow up analyses. Two identification methods, similarity coefficient-based and biomarker-based methods, are presented. Neuroscience A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research Brian H. Smith1, Christina M. Burden1 1School of Life Sciences, Arizona State University The Proboscis Extension Response (PER) conditioning protocol, developed for the honey bee (Apis mellifera), provides an ecologically-relevant and easily quantifiable means for studying several different mechanisms of learning in many insect species. Bioengineering Oscillation and Reaction Board Techniques for Estimating Inertial Properties of a Below-knee Prosthesis Jeremy D. Smith1, Abbie E. Ferris1, Gary D. Heise1, Richard N. Hinrichs2, Philip E. Martin3 1School of Sport & Exercise Science, University of Northern Colorado, 2Kinesiology Program, Arizona State University, 3Department of Kinesiology, Iowa State University Body segmental inertial properties are required for inverse dynamics modeling. Using an oscillation and reaction board technique, inertial properties of below-knee prostheses were measured. Using direct measures of prosthesis inertia in the inverse dynamics model of the prosthetic leg resulted in lower magnitudes of resultant joint forces and moments. Medicine Collecting Saliva and Measuring Salivary Cortisol and Alpha-amylase in Frail Community Residing Older Adults via Family Caregivers Nancy A. Hodgson1, Douglas A. Granger2,3,4 1Center for Innovative Care in Aging, Johns Hopkins University School of Nursing, 2Institute for Interdisciplinary Salivary Bioscience Research, Arizona State University, 3Johns Hopkins University School of Nursing, 4Johns Hopkins University Bloomberg School of Public Health We demonstrate: (1) procedures for collection of salivary samples in cognitive impaired older adults by family caregivers in the home setting, (2) procedures for measuring stress activity via salivary cortisol and alpha amylase, and (3) representative profiles. Protocols that allow researchers to study stress-linked processes advance our understanding of biological sensitivity and susceptibility. Biology Obtaining Specimens with Slowed, Accelerated and Reversed Aging in the Honey Bee Model Daniel Münch1, Nicholas Baker2, Erik M.K. Rasmussen1, Ashish K. Shah1, Claus D. Kreibich1, Lars E. Heidem1, Gro V. Amdam1,2 1Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, 2School of Life Sciences, Arizona State University In honey bee workers, aging depends on social behaviors rather than on chronological age. Here we show how worker-types with very different aging patterns can be obtained and analyzed for cellular senescence. Neuroscience RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera) Ying Wang1, Nicholas Baker1, Gro V. Amdam1,2 1School of Life Sciences, Arizona State University, 2Department of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences In this protocol, we describe two strategies that simultaneously suppress two genes (double gene knockdown) in honey bees. Then we present how to use the proboscis extension response (PER) assay to study the effect of double gene knockdown on honey bee gustatory perception. Biology Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins Kahlin Leuzinger*1, Matthew Dent*1, Jonathan Hurtado1, Jake Stahnke1, Huafang Lai1, Xiaohong Zhou1, Qiang Chen1 1The College of Technology and Innovation, Center for Infectious Disease and Vaccinology, The Biodesign Institute, Arizona State University Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression. Neuroscience Preparation of Drosophila Central Neurons for in situ Patch Clamping Stefanie Ryglewski1, Carsten Duch1 1School of Life Sciences, Arizona State University In situ patch clamp recordings are used for electrophysiological characterization of neurons in intact circuitry. In the Drosophila genetic model patch clamping is difficult because the CNS is small and surrounded by a robust sheath. This article describes the procedure to remove the sheath and clean neurons for subsequent patch clamp recordings. Bioengineering High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA) Chetan Patel1, Jit Muthuswamy1 1School of Biological and Health Systems Engineering, Arizona State University The article details the protocol for site-specific transfection of scrambled sequence of siRNA in an adherent mammalian cell culture using a microelectrode array (MEA). Biology Two Types of Assays for Detecting Frog Sperm Chemoattraction Lindsey A. Burnett1, Nathan Tholl2, Douglas E. Chandler2 1Department of Animal Sciences, University of Illinois, Urbana-Champaign, 2School of Life Sciences, Arizona State University Eggs and the extracellular coatings around eggs frequently release peptides, proteins and small molecules that communicate with sperm to guide them to the egg thereby promoting fertilization. Using frog sperm we describe and compare two classes of assays used to detect sperm chemoattraction – sperm accumulation assays and sperm tracking assays.