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41 ARTICLES PUBLISHED IN JoVE

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Biology

Crystallization of Membrane Proteins in Lipidic Mesophases
Wei Liu 1, Vadim Cherezov 1
1Molecular Biology, The Scripps Research Institute

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

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Neuroscience

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice
Janet Alder 1, Wendy Fujioka 1, Jonathan Lifshitz 2,3, David P. Crockett 1, Smita Thakker-Varia 1
1Department of Neuroscience and Cell Biology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, 2Spinal Cord and Brain Injury Research Center, 3Department of Anatomy and Neurobiology, Department of Physical Medicine and Rehabilitation, University of Kentucky Chandler Medical Center

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

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Immunology and Infection

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model
Dimitrios N. Vatakis 1,2,3, Gregory C. Bristol 1,2, Sohn G. Kim 1,2, Bernard Levin 1,2, Wei Liu 4, Caius G. Radu 4, Scott G. Kitchen 1,2,3, Jerome A. Zack 1,2,5
1Department of Medicine, Division of Hematology-Oncology, David Geffen School of Medicine at UCLA, 2UCLA AIDS Institute, 3Eli & Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, 4Department of Medical and Molecular Pharmacology, David Geffen School of Medicine at UCLA, 5Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine at UCLA

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

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Neuroscience

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)
Ying Wang 1, Nicholas Baker 1, Gro V. Amdam 1,2
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.

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Biology

Obtaining Specimens with Slowed, Accelerated and Reversed Aging in the Honey Bee Model
Daniel Münch 1, Nicholas Baker 2, Erik M.K. Rasmussen 1, Ashish K. Shah 1, Claus D. Kreibich 1, Lars E. Heidem 1, Gro V. Amdam 1,2
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.

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JoVE Core

Collecting Saliva and Measuring Salivary Cortisol and Alpha-amylase in Frail Community Residing Older Adults via Family Caregivers
Nancy A. Hodgson 1, Douglas A. Granger 2,3,4
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.

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Education

Oscillation and Reaction Board Techniques for Estimating Inertial Properties of a Below-knee Prosthesis
Jeremy D. Smith 1, Abbie E. Ferris 1, Gary D. Heise 1, Richard N. Hinrichs 2, Philip E. Martin 3
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.

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Neuroscience

A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research
Brian H. Smith 1, Christina M. Burden 1
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.

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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 Zhang 1, Katleen Vranckx 2, Koen Janssens 2, Todd R. Sandrin 1
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.

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Neuroscience

Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons
Megan N. Evilsizor 1,2, Helen F. Ray-Jones 1,3, Jonathan Lifshitz 1,2,4,5, Jenna Ziebell 1,2
1Department of Child Health, University of Arizona College of Medicine - Phoenix, 2BARROW Neurological Institute, Phoenix Children's Hospital, 3Department of Biology and Biochemistry, University of Bath, 4Neuroscience Program, Arizona State University, 5Phoenix VA Healthcare System

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.

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Bioengineering

Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis
Stephanie M. Williams 1, Lalitha Venkataraman 1, Huilai Tian 1, Galam Khan 2, Brent T. Harris 2,3, Michael R. Sierks 1
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.

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Biology

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay
Justin M. Wolter 1,2, Kasuen Kotagama 1,2, Cody S. Babb 2, Marco Mangone 1,2
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.

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Chemistry

Glycan Node Analysis: A Bottom-up Approach to Glycomics
Sahba Zaare 1, Jesús S. Aguilar 1, Yueming Hu 1, Shadi Ferdosi 1, Chad R. Borges 1
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.

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Biochemistry

Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography
Andrii Ishchenko 1,2, Vadim Cherezov 1,2, Wei Liu 3
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.

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Behavior

Assessing Working Memory in Children: The Comprehensive Assessment Battery for Children – Working Memory (CABC-WM)
Kathryn Cabbage 1, Shara Brinkley 2, Shelley Gray 2, Mary Alt 3, Nelson Cowan 4, Samuel Green 5, Trudy Kuo 6, Tiffany P. Hogan 1
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.

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Bioengineering

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening
Yaqian Li *1,2, Xiaojun Yan *1, Wei Liu *1, Lyu Zhou 1,3, Zhifeng You 1, Yanan Du 1,2
1Department of Biomedical Engineering, School of Medicine, Tsinghua University, 2Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, 3School of Life Sciences, Tsinghua University

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

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Chemistry

Quantifying X-Ray Fluorescence Data Using MAPS
Tara Nietzold 1, Bradley M. West 2, Michael Stuckelberger 2, Barry Lai 3, Stefan Vogt 3, Mariana I Bertoni 2
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.

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Neuroscience

SwarmSight: Real-time Tracking of Insect Antenna Movements and Proboscis Extension Reflex Using a Common Preparation and Conventional Hardware
Justas Birgiolas 1, Christopher M. Jernigan 1, Richard C. Gerkin 1, Brian H. Smith 1, Sharon M. Crook 1,2
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.

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Immunology and Infection

Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion
James J. Faust 1,2, Wayne Christenson 3,4,5, Kyle Doudrick 6, John Heddleston 7, Teng-Leong Chew 7, Marko Lampe 8, Arnat Balabiyev 1,2, Robert Ros 3,4,5, Tatiana P. Ugarova 1,2
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.

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Genetics

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms
Clark Fritsch 1,2, Jean-Francois Pierre Gout 3,4, Marc Vermulst 1
1Center for Mitochondrial and Epigenomic Medicine, Children's Hospital of Philadelphia, 2Department of Cellular and Molecular Biology, University of Pennsylvania, 3Department of Biological Sciences, Mississippi State University, 4Center for Mechanisms of Evolution, Biodesign Institute, Arizona State University

This protocol provides researchers with a new tool to monitor the fidelity of transcription in multiple model organisms.

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Engineering

Developing High Performance GaP/Si Heterojunction Solar Cells
Chaomin Zhang 1, Ehsan Vadiee 1, Som Dahal 1, Richard R. King 1, Christiana B. Honsberg 1
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.

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Environment

A Precise and Autonomous System for the Detection of Insect Emergence Patterns
Meghan M. Bennett 1,2, Joseph P. Rinehart 3, George D. Yocum 3, Ian Yocum 3
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.

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Neuroscience

Simultaneous Cryosectioning of Multiple Rodent Brains
Tabitha R.F. Green 1,2,3, J. Bryce Ortiz 1, Jordan L. Harrison 5, Jonathan Lifshitz 1,3,4, Rachel K. Rowe 1,3,4
1Department of Child Health, University of Arizona College of Medicine, 2Department of Biological Sciences, University of Bath, 3BARROW Neurological Institute at Phoenix Children's Hospital, 4Phoenix Veteran Affairs Healthcare System, 5Department of Basic Medical Sciences, University of Arizona College of Medicine

Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

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Immunology and Infection

Merkel Cell Polyomavirus Infection and Detection
Wei Liu *1, Nathan A. Krump *1, Christopher B. Buck 2, Jianxin You 1
1Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, 2Laboratory of Cellular Oncology, National Cancer Institute

Here, we present a protocol to infect primary human dermal fibroblast with MCPyV. The protocol includes isolation of dermal fibroblasts, preparation of MCPyV virions, virus infection, immunofluorescence staining, and fluorescence in situ hybridization. This protocol can be extended for characterizing MCPyV-host interactions and discovering other cell types infectable by MCPyV.

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Bioengineering

Using Nanoplasmon-Enhanced Scattering and Low-Magnification Microscope Imaging to Quantify Tumor-Derived Exosomes
Meihua Wan 1,2, Pouya Amrollahi 2,3, Dali Sun 4, Christopher Lyon 2,3, Tony Y. Hu 2,3
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.

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Biochemistry

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays
Fernanda Festa 1,2, Joshua Labaer 1
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.

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Neuroscience

Anti-RDL and Anti-mGlutR1 Receptors Antibody Testing in Honeybee Brain Sections using CRISPR-Cas9
Irina Sinakevitch 1,2, Zev Kurtzman *2, Hyun G. Choi *2, David Arturo Ruiz Pardo 3, Romain A. Dahan 2, Nathaniel Klein 1, Branimir Bugarija 4, Erik Wendlandt 4, Brian H. Smith 2
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.

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JoVE Core

X-ray Beam Induced Current Measurements for Multi-Modal X-ray Microscopy of Solar Cells
Christina Ossig 1,3, Tara Nietzold 2, Bradley West 2, Mariana Bertoni 2, Gerald Falkenberg 1, Christian G. Schroer 1,3, Michael E. Stuckelberger 1
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.

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Immunology and Infection

Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics (DCAF)
Xue Bai *1,2, Lin Zhang *3, Jin Hu 2, Xiuxiu Zhao 2, Jinheng Pan 2, Haiteng Deng 3, Shan Feng 1,2
1Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, 2Mass Spectrometry Core Facility, School of Life Sciences, Westlake University, 3MOE Key Laboratory of Bioinformatics, Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University

Development of a dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF) is novel for the elimination of harmful antibodies. Here, we describe a detailed protocol for the synthesis of DCAF1 molecule, which can selectively block 4G2 antibody to eliminate antibody dependent enhancement effect during Dengue virus infection.

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Bioengineering

Ovarian Cancer Detection Using Photoacoustic Flow Cytometry
Joel F. Lusk 1, Christopher Miranda 1, Barbara S. Smith 1
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.

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Genetics

A Screening Method for Identification of Heterochromatin-Promoting Drugs Using Drosophila
Lin Zhang 1, Kenny Dao 1, Angela Kang 1, Andre C. Loyola 1, Robin Shang 1, Jinghong Li 1, Willis X. Li 1
1Department of Medicine, University of California San Diego

Drosophila is a widely used experimental model suitable for screening drugs with potential applications for cancer therapy. Here, we describe the use of Drosophila variegated eye color phenotypes as a method for screening small-molecule compounds that promote heterochromatin formation.

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Engineering

Metal-Assisted Electrochemical Nanoimprinting of Porous and Solid Silicon Wafers
Aliaksandr Sharstniou 1, Stanislau Niauzorau 1, Ashlesha Junghare 1, Bruno P. Azeredo 1
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.

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Chemistry

Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay
Jesús Aguilar Diaz De Leon 1, 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.

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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. Hunter 1,2,3, Andrew G. Diamos 1,2,4, Boyd Armer 2,3, Tsafrir S. Mor 1,2,3, Hugh S. Mason 1,2,3
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.

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Bioengineering

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform
Jaimeson Veldhuizen 1, Mehdi Nikkhah 1,2
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.

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Biochemistry

Sample Preparation using a Lipid Monolayer Method for Electron Crystallographic Studies
Chloe D. Truong 1,2, Dewight R. Williams 3, Mary Zhu 1, Joseph Che-Yen Wang 4, Po-Lin Chiu 1,2
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.

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Genetics

Optimized Bone Sampling Protocols for the Retrieval of Ancient DNA from Archaeological Remains
Cody E. Parker 1,2, Kirsten I. Bos 1,3, Wolfgang Haak 1,3, Johannes Krause 1,3
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).

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Biochemistry

Purification of Endogenous Drosophila Transient Receptor Potential Channels
Jia Liu *1, Yuyang Liu *1, Weidi Chen 1, Yuzhen Ding 1, Xiaoru Lan 1, Wei Liu 1
1Shenzhen Key Laboratory for Neuronal Structural Biology, Biomedical Research Institute, Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center

Based on the assembling mechanism of the INAD protein complex, in this protocol, a modified affinity purification plus competition strategy was developed to purify the endogenous Drosophila TRP channel.

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Biology

Topical Application Bioassay to Quantify Insecticide Toxicity for Mosquitoes and Fruit Flies
Brook M. Jensen 1, Rachel A. Althoff 1, Sarah E. Rydberg 1, Emma N. Royster 1, Alden Estep 2, Silvie Huijben 1
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.

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Biology

Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis
Jinxi Yuan 1, Jie Zhang 1, Yan Zhang 1, WuYun QiQiGe 1, Wei Liu 2, Shanchun Yan 1, Guirong Wang 2
1Key Laboratory of Sustainable Forest Ecosystem Management - Ministry of Education, Northeast Forestry University, 2Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences

This paper presents the step-by-step protocols for CRISPR/Cas9 mutagenesis of the Oriental fruit fly Bactrocera dorsalis. Detailed steps provided by this standardized protocol will serve as a useful guide for generating mutant flies for functional gene studies in B. dorsalis.

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Biochemistry

Purification of Active Photosystem I-Light Harvesting Complex I from Plant Tissues
Christopher Gorski 1,2, Yuval Mazor 1,2
1School of Molecular Sciences, Arizona State University, 2Biodesign Center for Applied Structural Discovery, Arizona State University

This protocol describes the isolation of Photosystem I (PSI) - Light Harvesting Complex I (LHCI) from plant tissues. PSI along with PSII is responsible for the conversion of light to chemical energy in oxygenic photoautotrophs and has a quantum efficiency of ~1, making it a target for studying light-driven energy transfer.

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