Iowa State University View Institution's Website 35 articles published in JoVE Immunology and Infection Canine Intestinal Organoids in a Dual-Chamber Permeable Support System Vojtech Gabriel1, Christopher Zdyrski1, Dipak K. Sahoo2, Kimberly Dao3, Agnes Bourgois-Mochel2, Todd Atherly3, Marilyn N. Martinez4, Donna A. Volpe5, Jamie Kopper2, Karin Allenspach2,3, Jonathan P. Mochel1,3 1Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, 2Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Iowa State University, 33D Health Solutions Inc., 4Office of New Animal Drug Evaluation, Center for Veterinary Medicine, Food and Drug Administration, 5Division of Applied Regulatory Science, Office of Clinical Pharmacology, Office of Translational Sciences, Center for Drug Evaluation and Research, Food and Drug Administration Here, we present a protocol describing the culture of canine intestinal organoids in a dual-chamber, permeable support system. Organoid seeding in the permeable supports, the monolayer maintenance, and subsequent drug permeability experiments are described. Medicine Standardization and Maintenance of 3D Canine Hepatic and Intestinal Organoid Cultures for Use in Biomedical Research Vojtech Gabriel1, Christopher Zdyrski1, Dipak K. Sahoo2, Kimberly Dao3, Agnes Bourgois-Mochel2, Jamie Kopper2, Xi-Lei Zeng4, Mary K. Estes4, Jonathan P. Mochel1,3, Karin Allenspach2,3 1Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, 2Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Iowa State University, 33D Health Solutions Inc., 4Department of Molecular Virology and Microbiology, Baylor College of Medicine Experimental methods to harvest adult stem cells from canine intestinal and hepatic tissues to establish 3D organoid cultures are described. Furthermore, the laboratory techniques to ensure consistent growth and provide standard operating procedures to harvest, biobank, and revive canine intestinal and hepatic organoid cultures are discussed. Biochemistry High-Pressure NMR Experiments for Detecting Protein Low-Lying Conformational States Trang T. Nguyen1, Steven Siang2, Julien Roche2 1Department of Chemistry, Iowa State University, 2Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University We provide a detailed description of the steps required to assemble a high-pressure cell, set up and record high-pressure NMR experiments, and finally analyze both peak intensity and chemical shift changes under pressure. These experiments can provide valuable insights into the folding pathways and structural stability of proteins. Cancer Research Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1α Nanoparticles M. M. Hasibuzzaman1,2, Kathleen A. Ross3,4, Aliasger K. Salem1,4,5,6, Balaji Narasimhan3,4, Andrean L. Simons1,2,3,5,6,7 1Interdisciplinary Graduate Program in Human Toxicology, University of Iowa, 2Department of Pathology, University of Iowa, 3Department of Chemical and Biological Engineering, College of Engineering, Iowa State University, 4Nanovaccine Institute, Iowa State University, 5Division of Pharmaceutics and Translational Therapeutics, College of Pharmacy, University of Iowa, 6Holden Comprehensive Cancer Center, University of Iowa, 7Department of Oral Pathology, Radiology and Medicine, College of Dentistry, University of Iowa A standard protocol is described to study the antitumor activity and associated toxicity of IL-1α in a syngeneic mouse model of HNSCC. Bioengineering Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems Jared L. Dopp1, Nigel F. Reuel1 1Chemical and Biological Engineering, Iowa State University The study describes a protocol for creating large (µg-mg) quantities of DNA for protein screening campaigns from synthetic gene fragments without cloning or using living cells. The minimal template is enzymatically digested and circularized and then amplified using isothermal rolling circle amplification. Cell-free expression reactions could be performed with the unpurified product. Biochemistry 15N CPMG Relaxation Dispersion for the Investigation of Protein Conformational Dynamics on the µs-ms Timescale Aayushi Singh1, Jeffrey A. Purslow1, Vincenzo Venditti1,2 1Department of Chemistry, Iowa State University, 2Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University Here, a detailed description of the protocol implemented in the laboratory for acquisition and analysis of 15N relaxation dispersion profiles by solution NMR spectroscopy is provided. Biology Direct Agroinoculation of Maize Seedlings by Injection with Recombinant Foxtail Mosaic Virus and Sugarcane Mosaic Virus Infectious Clones Bliss M. Beernink*1, Katerina L. Holan*1, Ryan R. Lappe1, Steven A. Whitham1 1Department of Plant Pathology and Microbiology, Iowa State University An Agrobacterium-based injection (agroinjection) protocol is presented for the inoculation of foxtail mosaic virus and sugarcane mosaic virusclones into maize seedlings. Inoculation in this manner leads to viral infection, virus-induced gene silencing of marker genes, and viral overexpression of GFP. Biology Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes Alicia Masters1,2, Minjeong Kang1,3,4, Morgan McCaw1,3, Jacob D. Zobrist1,3,5, William Gordon-Kamm2, Todd Jones2, Kan Wang1,3 1Department of Agronomy, Iowa State University, 2Department of Applied Science and Technology, Corteva Agriscience, 3Crop Bioengineering Center, Iowa State University, 4Interdepartmental Plant Biology Major, Iowa State University, 5Interdepartmental Genetics and Genomics Major, Iowa State University Plant morphogenic genes can be used to improve genetic transformation of recalcitrant genotypes. Described here is an Agrobacterium-mediated genetic transformation (QuickCorn) protocol for three important public maize inbred lines. Behavior Using Flight Mills to Measure Flight Propensity and Performance of Western Corn Rootworm, Diabrotica virgifera virgifera (LeConte) Eric Yu Yu1, Aaron J. Gassmann1, Thomas W. Sappington2 1Department of Entomology, Iowa State University, 2Corn Insects & Crop Genetics Research Unit, USDA-ARS Flight mills are important tools for comparing how age, sex, mating status, temperature, or various other factors may influence an insect’s flight behavior. Here we describe protocols to tether and measure the flight propensity and performance of western corn rootworm under different treatments. Bioengineering Imaging Integrin Tension and Cellular Force at Submicron Resolution with an Integrative Tension Sensor Yuanchang Zhao1, Nathaniel M. Wetter1, Xuefeng Wang2 1Department of Physics and Astronomy, Iowa State University, 2Department of Physics and Astronomy, Molecular, Cellular, and Developmental Biology Interdepartmental Program, Iowa State University Integrin tension plays important roles in various cell functions. With an integrative tension sensor, integrin tension is calibrated with picoNewton (pN) sensitivity and imaged at submicron resolution. Biochemistry Analysis of Fucosylated Human Milk Trisaccharides in Biotechnological Context Using Genetically Encoded Biosensors Fatima Enam1, Thomas J. Mansell1 1Department of Chemical and Biological Engineering, Iowa State University We describe here the high-throughput detection and quantification of fucosylated human milk oligosaccharides (HMOs) using a whole-cell biosensor. We also demonstrate here, the adaptation of this platform towards analysis of HMO production strains, focusing on improving the signal to noise ratio. Medicine Evaluation of Stem Cell Therapies in a Bilateral Patellar Tendon Injury Model in Rats John R. Wagner1, Takashi Taguchi1, Jane Y. Cho2, Chandrashekhar Charavaryamath3, Dominique J. Griffon1 1College of Veterinary Medicine, Western University of Health Sciences, 2Applied Medical, 3College of Veterinary Medicine, Iowa State University This paper describes the preparation and evaluation of umbilical cord matrix-derived mesenchymal stem cells spheroids with a bilateral patellar tendon defect model in a rat. This model was associated with an acceptable morbidity and was found to detect differences between untreated and treated tendons, and between the two treatments tested. Developmental Biology A Modified Trier Social Stress Test for Vulnerable Mexican American Adolescents Megan M. Johnson1, Julianna Deardorff1, Kimberly Parra1, Abbey Alkon2, Brenda Eskenazi1, Elizabeth Shirtcliff3 1Center for Environmental Research and Children's Health (CERCH), Berkley School of Public Health, University of California, Berkeley, 2San Francisco (UCSF) School of Nursing, University of California, San Francisco, 3Human Development and Family Studies, Iowa State University Here, we present a protocol that provoked cortisol reactivity in a vulnerable adolescent Mexican American sample utilizing a modified version of the Trier Social Stress Test (TSST). Saliva samples were collected at baseline, 15, 30, and 45 min post-TSST onset. Future research could utilize this modified TSST with vulnerable youth. Neuroscience Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells Lauren A. Laboissonniere1, Takuma Sonoda2, Seul Ki Lee2, Jeffrey M. Trimarchi1, Tiffany M. Schmidt2 1Department of Genetics, Development, and Cell Biology, Iowa State University, 2Department of Neurobiology, Northwestern University Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity. Neuroscience Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility Souvarish Sarkar*1, Emir Malovic*1, Brandon Plante1, Gary Zenitsky1, Huajun Jin1, Vellareddy Anantharam1, Arthi Kanthasamy1, Anumantha G. Kanthasamy1 1Biomedical Sciences & Iowa Center for Advanced Neurotoxicology, Iowa State University Here, we present a protocol to isolate microglia from postnatal mouse pups (day 1) for in vitro experimentation. This improvised method of isolation generates both high yield and purity, a significant advantage over alternate methods that allows broad range experimentation for the purposes of elucidating microglial biology. Immunology and Infection A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release Unity Jeffery1, Robert D. Gray2, Dana N. LeVine3 1Department of Veterinary Microbiology and Preventative Medicine, College of Veterinary Medicine, Iowa State University, 2MRC Centre for Inflammation Research, University of Edinburgh, 3Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Iowa State University Neutrophil extracellular traps (NETs) are networks of DNA, histones and neutrophil proteins. Although a component of the innate immune response, NETs are implicated in autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay. Environment Laboratory Simulation of an Iron(II)-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria Markus Maisch1,2, Wenfang Wu1, Andreas Kappler1, Elizabeth D. Swanner1,2 1Department of Geosciences, University of Tuebingen, 2Department of Geological and Atmospheric Sciences, Iowa State University We simulated a Precambrian ferruginous marine upwelling system in a lab-scale vertical flow-through column. The goal was to understand how geochemical profiles of O2 and Fe(II) evolve as cyanobacteria produce O2. The results show the establishment of a chemocline due to Fe(II) oxidation by photosynthetically produced O2. Biology Genome Editing in Astyanax mexicanus Using Transcription Activator-like Effector Nucleases (TALENs) Johanna E. Kowalko1, Li Ma2, William R. Jeffery3 1Genetics, Development and Cell Biology, Iowa State University, 2Department of Biological Sciences, University of Cincinnati, 3Department of Biology, University of Maryland Gene-targeting mutagenesis is now possible in a wide range of organisms using genome editing techniques. Here, we demonstrate a protocol for targeted gene mutagenesis using transcription activator like effector nucleases (TALENs) in Astyanax mexicanus, a species of fish that includes surface fish and cavefish. Biology High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension Ganesh P. Subedi1, Roy W. Johnson2, Heather A. Moniz2, Kelley W. Moremen2, Adam W. Barb1 1The Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, 2Complex Carbohydrate Research Center, University of Georgia Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition. Immunology and Infection Application of Long-term cultured Interferon-γ Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle Mayara F. Maggioli1,2, Mitchell V. Palmer1, H. Martin Vordermeier3, Adam O. Whelan3, James M. Fosse4, Brian J. Nonnecke1, W. Ray Waters1 1Infectious Bacterial Diseases Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, 2Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University, 3UK Veterinary Laboratories Agency, 4Visual Services, National Centers for Animal Health, Animal and Plant Health Inspection Service, United States Department of Agriculture Long-term cultured interferon-γ enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells. Medicine High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies Anup D. Sharma1, Pavel A. Brodskiy1,3, Emma M. Petersen2,3, Melih Dagdeviren2, Eun-Ah Ye2, Surya K. Mallapragada1, Donald Sakaguchi2,3 1Department of Chemical and Biological Engineering, Iowa State University, 2Department of Genetics, Development and Cell Biology, Iowa State University, 3Biology Program, Iowa State University This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair. 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. Engineering Characterization of Thermal Transport in One-dimensional Solid Materials Guoqing Liu1, Huan Lin1, Xiaoduan Tang1, Kevin Bergler1, Xinwei Wang1 1Department of Mechanical Engineering, Iowa State University The TET (transient electro-thermal) technique is an effective approach developed to measure the thermal diffusivity of solid materials. Engineering High-resolution, High-speed, Three-dimensional Video Imaging with Digital Fringe Projection Techniques Laura Ekstrand1, Nikolaus Karpinsky1, Yajun Wang1, Song Zhang1 13D Machine Vision Laboratory, Department of Mechanical Engineering, Iowa State University This video describes the fundamentals of digital fringe projection techniques, which provide dense 3D measurements of dynamically changing surfaces. It also demonstrates the design and operation of a high-speed binary defocusing system based on these techniques. Biology Spatial Multiobjective Optimization of Agricultural Conservation Practices using a SWAT Model and an Evolutionary Algorithm Sergey Rabotyagov1, Todd Campbell2, Adriana Valcu2, Philip Gassman2, Manoj Jha3, Keith Schilling4, Calvin Wolter4, Catherine Kling2 1School of Environmental and Forest Sciences, University of Washington, 2Center for Agricultural and Rural Development, Department of Economics, Iowa State University, 3Department of Civil, Architectural, and Environmental Engineering, North Carolina A&T University, 4Iowa Geological and Water Survey This work demonstrates an integration of a water quality model with an optimization component utilizing evolutionary algorithms to solve for optimal (lowest-cost) placement of agricultural conservation practices for a specified set of water quality improvement objectives. The solutions are generated using a multi-objective approach, allowing for explicit quantification of tradeoffs. Bioengineering Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries Latrisha K. Petersen1, Ana V. Chavez-Santoscoy1, Balaji Narasimhan1 1Department of Chemical and Biological Engineering, Iowa State University This method describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. Bioengineering High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles Brenda R. Carrillo-Conde*1, Rajarshi Roychoudhury*2, Ana V. Chavez-Santoscoy*1, Balaji Narasimhan1, Nicola L.B. Pohl1,2 1Department of Chemical and Biological Engineering, Iowa State University, 2Department of Chemistry, Iowa State University In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells. Bioengineering Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry Yashdeep Phanse1, Amanda E. Ramer-Tait1, Sherree L. Friend2, Brenda Carrillo-Conde3, Paul Lueth1, Carrie J. Oster1, Gregory J. Phillips1, Balaji Narasimhan3, Michael J. Wannemuehler1, Bryan H. Bellaire1 1Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, 2Amnis Corporation, 3Department of Chemical and Biological Engineering, Iowa State University In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells. Bioengineering Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles Ana V. Chavez-Santoscoy1, Lucas M. Huntimer2, Amanda E. Ramer-Tait2, Michael Wannemuehler2, Balaji Narasimhan1 1Department of Chemical and Biological Engineering, Iowa State University, 2Department of Veterinary Microbiology and Preventive Medicine, Iowa State University Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles. Neuroscience Single-cell Profiling of Developing and Mature Retinal Neurons Jillian J. Goetz1, Jeffrey M. Trimarchi1 1Department of Genetics, Development and Cell Biology, Neuroscience Program, Iowa State University A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types. Biology Cryopreservation of Preimplantation Embryos of Cattle, Sheep, and Goats Curtis R. Youngs1 1Animal Science Department, Iowa State University Preimplantation embryos may be cryopreserved after placement into a hypertonic cryoprotective solution to cause cellular dehydration. After equilibration, ice crystal formation is induced in the solution surrounding the embryo. Further dehydration occurs as the embryo is slowly cooled to subzero temperatures before plunging into liquid nitrogen for storage. Immunology and Infection Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce Bledar Bisha1, Byron F. Brehm-Stecher2 1Center for Meat Safety and Quality, Department of Animal Sciences, Colorado State University, 2Rapid Microbial Detection and Control Laboratory, Department of Food Science and Human Nutrition, Iowa State University This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH). Biology Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling Weili Cai1, Ye Jin1, Jack Girton1, Jorgen Johansen1, Kristen M. Johansen1 1Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling. Biology Protocols for Oral Infection of Lepidopteran Larvae with Baculovirus Wendy Sparks1, Huarong Li1, Bryony Bonning1 1Department of Entomology, Iowa State University In this video, we demonstrate oral infection techniques of lepidopteran larvae with baculovirus in order to determine insecticidal efficiency. Biology Protocols for Microapplicator-assisted Infection of Lepidopteran Larvae with Baculovirus Huarong Li1, Wendy Sparks1, Bryony Bonning1 1Department of Entomology, Iowa State University In this video, we demonstrate two microapplicator techniques used to infect of lepidopteran larvae with baculovirus in order to determine insecticidal efficiency.