Pennsylvania State University View Institution's Website 37 articles published in JoVE Medicine Intradermal Microdialysis: An Approach to Investigating Novel Mechanisms of Microvascular Dysfunction in Humans Auni C. Williams*1, Virginia G. Content*1, Nathalie V. Kirby1, Lacy M. Alexander1 1Department of Kinesiology, The Pennsylvania State University Intradermal microdialysis is a minimally invasive technique used to investigate microvascular function in health and disease. Both dose-response and local heating protocols can be utilized for this technique to explore mechanisms of vasodilation and vasoconstriction in the cutaneous circulation. Engineering Measuring Local Tissue Strains in Tendons via Open-Source Digital Image Correlation Stanton Godshall1, Krishna Pedaprolu1, Erica Vasti1, Faezeh Eskandari2, Spencer E. Szczesny1,3 1Department of Biomedical Engineering, Pennsylvania State University, 2Department of Biomedical Engineering, Amirkabir University of Technology, 3Department of Orthopaedics and Rehabilitation, Pennsylvania State University This paper describes an open-source digital image correlation algorithm for measuring local 2D tissue strains within tendon explants. The accuracy of the technique has been validated using multiple techniques, and it is available for public use. Cancer Research Intra-Peritoneal Transplantation for Generating Acute Myeloid Leukemia in Mice Fenghua Qian1, Brooke E. Arner1, Shaneice K. Nettleford1, Robert F. Paulson1, K. Sandeep Prabhu1 1Department of Veterinary and Biomedical Sciences, Center for Molecular Immunology and Infectious Disease and Center for Molecular Toxicology and Carcinogenesis, Penn State Cancer Institute, The Pennsylvania State University Here, intra-peritoneal injection of leukemia cells is utilized to establish and propagate acute myeloid leukemia (AML) in mice. This new method is effective in the serial transplantation of AML cells and can serve as an alternative for those who may experience difficulties and inconsistencies with intravenous injection in mice. Bioengineering Gelatin Methacryloyl Granular Hydrogel Scaffolds: High-throughput Microgel Fabrication, Lyophilization, Chemical Assembly, and 3D Bioprinting Zaman Ataie1, Arian Jaberi1, Sina Kheirabadi1, Aneesh Risbud2, Amir Sheikhi1,2 1Department of Chemical Engineering, The Pennsylvania State University, 2Department of Biomedical Engineering, The Pennsylvania State University This article describes protocols for high-throughput gelatin methacryloyl microgel fabrication using microfluidic devices, converting microgels to resuspendable powder (micro-aerogels), the chemical assembly of microgels to form granular hydrogel scaffolds, and developing granular hydrogel bioinks with preserved microporosity for 3D bioprinting. Biochemistry Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice Liza-Anastasia DiCecco*1,2, Samantha Berry*1, G. M. Jonaid*1,3, Maria J. Solares1,4, Liam Kaylor1,4, Jennifer L. Gray5, Carol Bator6, William J. Dearnaley1, Michael Spilman7, Madeline J. Dressel-Dukes8, Kathryn Grandfield2, Sarah M. McDonald Esstman9, Deborah F. Kelly1,5,6 1Department of Biomedical Engineering, Pennsylvania State University, 2Department of Materials Science and Engineering, McMaster University, 3Bioinformatics and Genomics Graduate Program, Huck Institutes of the Life Sciences, Pennsylvania State University, 4Molecular, Cellular, and Integrative Biosciences Graduate Program, Huck Institutes of the Life Sciences, Pennsylvania State University, 5Materials Research Institute, Pennsylvania State University, 6Huck Institutes of the Life Sciences, Pennsylvania State University, 7Applications team, Direct Electron, 8Application Scientist, Protochips, Inc., 9Department of Biology, Wake Forest University Here protocols are described to prepare virus assemblies suitable for liquid-EM and cryo-EM analysis at the nanoscale using transmission electron microscopy. Neuroscience Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media Madison T. Jones1, Samantha W. Manioci1, Ashley E. Russell1,2 1Department of Biology, School of Science, Penn State Erie, The Behrend College, 2Magee Womens Research Institute—Allied Member The protocol here demonstrates that extracellular vesicles can be adequately separated from conditioned cell culture media using size exclusion chromatography. 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. Environment The Role of Fabric in Frictional Properties of Phyllosilicate-Rich Tectonic Faults Cristiano Collettini1,4, Telemaco Tesei2, Fabio Trippetta1, Marco M. Scuderi1, Eliza Richardson3, Chris Marone1,3, Giacomo Pozzi4, Cecilia Viti5 1Dipartimento di Scienze della Terra, Sapienza Università di Roma, 2Dipartimento di Geoscienze, Università di Padova, 3Department of Geoscience, The Pennsylvania State University, 4Istituto Nazionale di Geofisica e Vulcanologia, 5Dipartimento di Scienze Fisiche, della Terra e dell’Ambiente, Università di Siena Friction of phyllosilicates-rich faults sheared in their in situ geometry is significantly lower than friction of their powdered equivalents. Bioengineering Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation Niloufar Fattahi1, Niloy Barua1, André J. van der Vlies2, Ryan R. Hansen1 1Tim Taylor Department of Chemical Engineering, Kansas State University, 2Department of Materials Science and Engineering, The Pennsylvania State University The use of photodegradable hydrogels to isolate bacterial cells by utilizing a high-resolution light pattering tool is reported. Essential experimental procedures, results, and advantages of the process are reviewed. The method enables rapid and inexpensive isolation of targeted bacteria showing rare or unique functions from heterogeneous communities or populations. Chemistry Picometer-Precision Atomic Position Tracking through Electron Microscopy Leixin Miao1, Adrian Chmielewski1, Debangshu Mukherjee2, Nasim Alem1 1Department of Materials Science & Engineering, The Pennsylvania State University, 2Center for Nanophase Materials Sciences, Oak Ridge National Laboratory This work presents a workflow for atomic position tracking in atomic resolution transmission electron microscopy imaging. This workflow is performed using an open-source Matlab app (EASY-STEM). Biology Pupal and Adult Injections for RNAi and CRISPR Gene Editing in Nasonia vitripennis Elena Dalla Benetta*1, Duverney Chaverra-Rodriguez*1, Jason L. Rasgon2, Omar S. Akbari1 1Division of Biological Sciences, Section of Cell and Developmental Biology, University of California, San Diego, 2Department of Entomology, The Center for Infectious Disease Dynamics, The Huck Institutes of the Life Sciences, The Pennsylvania State University Here, we describe methods for efficient pupal and adult injections in Nasonia vitripennis as accessible alternatives to embryo microinjection, enabling functional analysis of genes of interest using either RNA-silencing via RNA interference (RNAi) or gene knockout via CRISPR/Cas9 genome editing. Behavior A Standardized Protocol for Preference Testing to Assess Fish Welfare Cairsty DePasquale1, Jennifer Sturgill1, Victoria A. Braithwaite2 1Department of Biology, Pennsylvania State University - Altoona, 2Department of Ecosystem Science & Management, Pennsylvania State University A fundamental aspect of assessing the welfare of animals in captivity is to ask whether the animals have what they want. Here, we present a protocol to determine housing preference in the zebrafish (Danio rerio) with respect to the presence/absence of environmental enrichment and access to flowing of water. Behavior Assessment of Midline Lingual Point-Pressure Somatosensation Using Von Frey Hair Monofilaments Nicole M. Etter1, Scott P. Breen2,3, Maya I. M. Alcala2,3, Gregory R. Ziegler3, John E. Hayes2,3 1Department of Communication Sciences and Disorders, College of Health and Human Development, The Pennsylvania State University, 2Sensory Evaluation Center, The Pennsylvania State University, 3Department of Food Science, College of Agricultural Sciences, The Pennsylvania State University This work describes a standard method to assess tactile sensation at the midline of the tongue tip. Using Von Frey Hair (VFH) monofilaments, this protocol provides estimates of detection and discrimination threshold estimates for oral point pressure (OPP). 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. Immunology and Infection An Immunohistopathologic Study to Profile the Folate Receptor Beta Macrophage and Vascular Immune Microenvironment in Giant Cell Arteritis Shirley Albano-Aluquin1, Jozef Malysz2, Michal Kidacki3, Manohar Ratnam4, Nancy J. Olsen1 1Department of Medicine, Penn State University, 2Department of Pathology, Penn State University, 3Penn State College of Medicine, 4Department of Oncology, Wayne State University School of Medicine The protocol illustrates the use of histopathologic examination and immunohistochemistry to profile the folate receptor beta macrophage and its relationship with the total immune cell infiltrate in temporal artery biopsies in giant cell arteritis. Environment Wastewater Irrigation Impacts on Soil Hydraulic Conductivity: Coupled Field Sampling and Laboratory Determination of Saturated Hydraulic Conductivity Jack E. Watson1, Tyson Robb2, Danielle Andrews-Brown3, Melissa Miller1 1Department of Ecosystem Science and Management, Pennsylvania State University, 2Department of Geography and Environmental Sustainability, State University of New York, Oneonta, 3Department of Geology and Environmental Science, University of Pittsburgh Here we present a methodology which matches a soil sample size and a hydraulic conductivity measurement device to prevent the so-called wall flow along the inside of the soil container from being erroneously included in water flow measurements. Its use is demonstrated with samples collected from a wastewater irrigation site. Immunology and Infection Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry Krista M. Armbruster1, Timothy C. Meredith1 1Department of Biochemistry and Molecular Biology, Pennsylvania State University The enrichment of bacterial lipoproteins using a non-ionic surfactant phase partitioning method is described for direct use in TLR assays or other applications. Further steps are detailed to prepare N-terminal tryptic lipopeptides for structural characterization by mass spectrometry. Environment Continuous Hydrologic and Water Quality Monitoring of Vernal Ponds Odette Mina1,2, Heather E. Gall1, Joseph W. Chandler1, Jeremy Harper3, Malcolm Taylor1 1Department of Agricultural and Biological Engineering, Pennsylvania State University, 2Energy and Environmental Sustainability Laboratory, Pennsylvania State University, 3Department of Ecosystem Science and Management, Pennsylvania State University Understanding the ecosystem services and processes provided by vernal ponds and the impacts of anthropogenic activities on their ability to provide these services requires intensive hydrologic monitoring. This sampling protocol using in-situ monitoring equipment was developed to understand the impact of anthropogenic activities on water levels and quality. Chemistry Determination of Thermodynamic Properties of Alkaline Earth-liquid Metal Alloys Using the Electromotive Force Technique Thomas P. Nigl1, Nathan D. Smith1, Timothy Lichtenstein1, Jarrod Gesualdi1, Kuldeep Kumar1, Hojong Kim1 1Department of Materials Science and Engineering, The Pennsylvania State University This protocol describes the measurement of the electromotive force of alkaline-earth elements in liquid metal alloys at high temperatures (723-1,123 K) to determine their thermodynamic properties, including activity, partial molar entropy, partial molar enthalpy, and phase transition temperatures, over a wide composition range. Bioengineering PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions Djoshkun Shengjuler1, Simou Sun2, Paul S. Cremer1,2, Craig E. Cameron1 1Department of Biochemistry and Molecular Biology, The Pennsylvania State University, 2Department of Chemistry, The Pennsylvania State University Here we present a supported lipid bilayer in the context of a microfluidic platform to study protein-phosphoinositide interactions using a label-free method based on pH modulation. Immunology and Infection Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation Joselyn N. Allen1, Adwitia Dey1, Ruth Nissly2, James Fraser1, Shan Yu1, Gayathri Balandaram1, Jeffrey M. Peters1, Pamela A. Hankey-Giblin1 1Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, 2Microscopy and Cytometry Facility, The Huck Institutes of Life Sciences, Pennsylvania State University This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders. Immunology and Infection Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response Antoine M. G. Barreaux1,2, Priscille Barreaux1,2, Matthew B. Thomas2, Jacob C. Koella1 1Laboratoire d'écologie et d'épidémioloigie parasitaire, Institut de biologie, Université de Neuchâtel, 2Merkle Lab, Department of Entomology, Pennsylvania State University Through inoculation with beads, the described technique enables the stimulation of the mosquito melanization response in the hemolymph circulating system. The amount of melanin covering the beads can be measured after dissection as a measure of the immune response. Engineering Characterization of Full Set Material Constants and Their Temperature Dependence for Piezoelectric Materials Using Resonant Ultrasound Spectroscopy Liguo Tang1, Wenwu Cao2 1Key Laboratory of Underwater Acoustic Communication and Marine Information Technology, Xiamen University, 2Department of Mathematics and Material Research Institute, The Pennsylvania State University This protocol describes the procedure of measuring the temperature dependence of the full set material constants of piezoelectric materials using resonant ultrasound spectroscopy (RUS). Developmental Biology Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line Mackenzie M. Shipley1, Colleen A. Mangold1, Moriah L. Szpara1 1Department of Biochemistry and Molecular Biology, The Huck Institutes of the Life Sciences, The Pennsylvania State University It is critical in neurobiology and neurovirology to have a reliable, replicable in vitro system that serves as a translational model for what occurs in vivo in human neurons. This protocol describes how to culture and differentiate SH-SY5Y human neuroblastoma cells into viable neurons for use in in vitro applications. Medicine Povidone Iodine Rectal Preparation at Time of Prostate Needle Biopsy is a Simple and Reproducible Means to Reduce Risk of Procedural Infection Jay D. Raman1, Kathleen K. Lehman1, Kalyan Dewan2, Girish Kirimanjeswara2 1Division of Urology, Penn State Milton S. Hershey Medical Center, 2Department of Veterinary and Biomedical Sciences, Penn State University Here, we present a protocol for use at the time of transrectal ultrasound guided prostate needle biopsy (TRUS PNB) that is a simple and cost-effective means to reduce infections following the procedure. Environment The Use of an Automated System (GreenFeed) to Monitor Enteric Methane and Carbon Dioxide Emissions from Ruminant Animals Alexander N. Hristov1, Joonpyo Oh1, Fabio Giallongo1, Tyler Frederick1, Holley Weeks1, Patrick R. Zimmerman2, Michael T. Harper1, Rada A. Hristova3, R. Scott Zimmerman2, Antonio F. Branco4 1Department of Animal Science, Pennsylvania State University, 2C-Lock, Inc. Rapid City, SD, 3Color Productions, State College, PA, 4Departamento de Zootecnia, Universidade Estadual de Maringá Accuracy and precision of the techniques used to measure methane emissions from ruminant animals are critically important for the success of greenhouse gas mitigation efforts. This manuscript describes the principles and operation of an automated system to monitor methane and carbon dioxide mass fluxes from the breath of ruminant animals. Behavior Applying Stereotactic Injection Technique to Study Genetic Effects on Animal Behaviors Colleen McSweeney1, Yingwei Mao1 1Department of Biology, The Pennsylvania State University Stereotactic injection of lentiviruses expressing cDNAs or shRNAs can modulate gene expression in specific brain areas of mice. Here, we present a protocol to combine stereotactic injections with behavioral tasks, such as the Open Field Test (OFT) and the Forced Swim Test (FST). Bioengineering Molecular Entanglement and Electrospinnability of Biopolymers Lingyan Kong1, Gregory R. Ziegler1 1Department of Food Science, Pennsylvania State University Electrospinning is a fascinating technique used to fabricate micro- to nano-scale fibers from a wide variety of materials. Molecular entanglement of the constituent polymers in the spinning dope is essential for successful electrospinning. We present a protocol for utilizing rheology to evaluate the electrospinnability of two biopolymers, starch and pullulan. Medicine Dried Blood Spot Collection of Health Biomarkers to Maximize Participation in Population Studies Michael W. Ostler1, James H. Porter1,2, Orfeu M. Buxton1,2,3,4 1Center for Population and Development Studies, Harvard School of Public Health, 2 Biomarkers are directly-measured biological indicators of disease or health. In population and social sciences, biomarkers need to be easy to obtain, transport, and analyze. Dried Blood Spot (DBS) collection meets this need, can be collected in the field with high response rates and analyzed for a variety of biomarkers. Immunology and Infection Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells Gregory Berry1, Hanspeter Waldner1 1Department of Microbiology & Immunology, Pennsylvania State University College of Medicine We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells. Engineering Encapsulation and Permeability Characteristics of Plasma Polymerized Hollow Particles Anaram Shahravan1, Themis Matsoukas1 1Department of Chemical Engineering, The Pennsylvania State University We have used plasma enhanced chemical vapor deposition to deposit thin films ranging from a few nm to several 100 nm on nano-sized particles of various materials. We subsequently etch the core material to produce hollow nanoshells whose permeability is controlled by the thickness of the shell. We characterize the permeability of these coatings to small solutes and demonstrate that these barriers can provide sustained release of the core material over several days. Immunology and Infection Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes Fengyang Lei1, Rizwanul Haque1, Xiaofang Xiong1, Jianxun Song1 1Department of Microbiology and Immunology, Pennsylvania State University College of Medicine Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes. Neuroscience A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain Andy Y. Shih1, Celine Mateo1, Patrick J. Drew2,3, Philbert S. Tsai1, David Kleinfeld1,4 1Department of Physics, University of California, San Diego, 2Department of Engineering Science and Mechanics, Pennsylvania State University, 3Department of Neurosurgery, Pennsylvania State University, 4Section of Neurobiology, University of California, San Diego We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy. Biology Isolation of CD133+ Liver Stem Cells for Clonal Expansion C. Bart Rountree1, Wei Ding1, Hein Dang1, Colleen VanKirk2, Gay M. Crooks3 1Department of Pediatrics and Pharmacology, Pennsylvania State College of Medicine, 2Department of Pharmacology, Pennsylvania State College of Medicine, 3Department of Pediatrics, University of California Los Angeles, School of Medicine Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion. Biology Primer-Free Aptamer Selection Using A Random DNA Library Weihua Pan1, Ping Xin1, Susan Patrick1, Stacey Dean2, Christine Keating2, Gary Clawson3,4 1Department of Pathology, Hershey Medical Center, Pennsylvania State University, 2Department of Chemistry, Pennsylvania State University, 3Departments of Pathology, and Biochemistry and Molecular Biology, Hershey Medical Center, Pennsylvania State University, 4Materials Research Institute, Pennsylvania State University SELEX protocols comprise multiple rounds of selection, each of which require regeneration of bound ligands, which in turn require fixed primer sequences flanking the random library regions. These fixed primer sequences can interfere with the selection process (false positives and negatives). Here we present a primer-free protocol. Neuroscience Low-stress Route Learning Using the Lashley III Maze in Mice Amanda Bressler1, David Blizard2, Anne Andrews3,4,5,6 1Department of Chemistry, Pennsylvania State University, 2Center for Developmental and Health Genetics, Pennsylvania State University, 3Department of Veterinary and Biomedical Sciences, Pennsylvania State University, 4Huck Institute of the Life Sciences, Pennsylvania State University, 5California NanoSystems Institute, University of California, Los Angeles, 6Semel Institute of Neuroscience and Human Behavior, University of California, Los Angeles The Lashley III maze is a route-learning task that does not rely on aversive stimuli or visual cues. It is thus a highly attractive option for evaluating learning and memory, especially in aging mice or otherwise where stress is a consideration. Biology Measuring Plant Cell Wall Extension (Creep) Induced by Acidic pH and by Alpha-Expansin Daniel M. Durachko1, Daniel J. Cosgrove1 1Department of Biology, Penn State University We demonstrate the use of a constant-force extensometer to measure long-term extension (creep) of plant cell wall specimens induced by acidic buffers and expansin protein.