University of Iowa View Institution's Website 33 articles published in JoVE Cancer Research Multiplexed Live-Cell Imaging for Drug Responses in Patient-Derived Organoid Models of Cancer Kaitriana E. Colling*1,2, Emily L. Symons*1, Lorenzo Buroni*3, Hiruni K. Sumanasiri1, Jessica Andrew-Udoh1, Emily Witt1,4, Haley A. Losh1, Abigail M. Morrison1, Kimberly K. Leslie5, Christopher J. Dunnill6, Johann S. de Bono3, Kristina W. Thiel1,7 1Department of Obstetrics and Gynecology, Carver College of Medicine, University of Iowa, 2Cancer Biology Graduate Program, Carver College of Medicine, University of Iowa, 3The Institute of Cancer Research: and the Royal Marsden NHS Foundation Trust, 4Department of Radiation Oncology, Carver College of Medicine, University of Iowa, 5Division of Molecular Medicine, Departments of Internal Medicine and Obstetrics and Gynecology, University of New Mexico Comprehensive Cancer Center, University of New Mexico Health Sciences Center, 6Agilent Technologies, 7Holden Comprehensive Cancer Center, University of Iowa Patient-derived tumor organoids are a sophisticated model system for basic and translational research. This methods article details the use of multiplexed fluorescent live-cell imaging for simultaneous kinetic assessment of different organoid phenotypes. Medicine Biobanking of Human Aqueous and Vitreous Liquid Biopsies for Molecular Analyses Julian Wolf1,2, Teja Chemudupati1,2, Aarushi Kumar1,2, Ditte K. Rasmussen1,2, Karen M. Wai2, Robert T. Chang2, Artis A. Montague2, Peter H. Tang3,4, Alexander G. Bassuk5,6,7, Antoine Dufour8,9, Prithvi Mruthrunjaya2, Vinit B. Mahajan1,2,10 1Molecular Surgery Laboratory, Stanford University, 2Department of Ophthalmology, Byers Eye Institute, Stanford University, 3Department of Ophthalmology and Visual Neurosciences, University of Minnesota, 4Retina Consultants of Minnesota, 5Department of Pediatrics, University of Iowa, 6Department of Neurology, University of Iowa, 7The Iowa Neuroscience Institute (INI), University of Iowa, 8Department of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, 9Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, 10Veterans Affairs Palo Alto Health Care System This protocol presents an integrated biorepository platform for the standardized collection, annotation, and biobanking of high-quality human aqueous humor and vitreous liquid biopsies for molecular downstream analyses, including proteomics, metabolomics, and glycomics. Genetics Mouse In Vivo Placental Targeted CRISPR Manipulation Annemarie J. Carver1,2,3, Robert J. Taylor2,3, Hanna E. Stevens1,2,3,4 1Interdisciplinary Graduate Program in Genetics, University of Iowa, 2Department of Psychiatry, Carver College of Medicine, University of Iowa, 3Iowa Neuroscience Institute, Carver College of Medicine, University of Iowa, 4Hawk-IDDRC, University of Iowa Here we describe a time-specific method to effectively manipulate critical developmental pathways in the mouse placenta in vivo. This is performed through the injection and electroporation of CRISPR plasmids into the placentas of pregnant dams on embryonic day 12.5. Immunology and Infection Use of In Vivo Imaging to Screen for Morphogenesis Phenotypes in Candida albicans Mutant Strains During Active Infection in a Mammalian Host Rohan S. Wakade1, Damian J. Krysan1,2, Melanie Wellington1 1Department of Pediatrics, Carver College of Medicine, University of Iowa, 2Department of Microbiology and Immunology, Carver College of Medicine, University of Iowa This manuscript describes a method for screening moderately sized Candida albicans mutant libraries for morphogenesis phenotypes during active infection in a mammalian host using non-invasive confocal microscopy. Neuroscience Modeling Human Cerebellar Development In Vitro in 2D Structure Deniz A. Madencioglu1,3,4,5, Karina A. Kruth1,3,4,5, Thomas H. Wassink1,3, Vincent A. Magnotta1,2,3,4,5, John A. Wemmie1,3,4,5, Aislinn J. Williams1,3,4,5 1Department of Psychiatry, University of Iowa, 2Department of Radiology, University of Iowa, 3Carver College of Medicine, University of Iowa, 4Iowa Neuroscience Institute, University of Iowa, 5Pappajohn Biomedical Institute, University of Iowa The present protocol explains the generation of a 2D monolayer of cerebellar cells from induced pluripotent stem cells for investigating the early stages of cerebellar development. Behavior Investigating Migraine-Like Behavior Using Light Aversion in Mice Mengya Wang1, Bianca N. Mason2, Levi P. Sowers3,4, Adisa Kuburas4, Brandon J. Rea3,4, Andrew F. Russo3,4,5 1Department of Neuroscience and Pharmacology, University of Iowa, 2School of Behavioral and Brain Sciences, University of Texas at Dallas, 3Center for the Prevention and Treatment of Visual Loss, Veterans Administration Health Center, Iowa City, IA, 4Department of Molecular Physiology and Biophysics, University of Iowa, 5Department of Neurology, University of Iowa Rodents are not able to report migraine symptoms. Here, we describe a manageable test paradigm (light/dark and open field assays) to measure light aversion, one of the most common and bothersome symptoms in patients with migraines. 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. Cancer Research Modeling the Effects of Hemodynamic Stress on Circulating Tumor Cells using a Syringe and Needle Devon L. Moose1,2, Sophia Williams-Perez3, Renee Cafun1, Benjamin L. Krog1, Michael D. Henry1,2,4,5,6 1Department of Molecular Physiology and Biophysics, Carver College of Medicine, University of Iowa, 2Holden Comprehensive Cancer Center, University of Iowa, 3MD program, Carver College of Medicine, University of Iowa, 4Department of Pathology, Carver College of Medicine, University of Iowa, 5Department of Urology, Carver College of Medicine, University of Iowa, 6Department of Radiation Oncology, Carver College of Medicine, University of Iowa Here we demonstrate a method to apply fluid shear stress to cancer cells in suspension to model the effects of hemodynamic stress on circulating tumor cells. Developmental Biology Murine Excisional Wound Healing Model and Histological Morphometric Wound Analysis Lindsey Rhea1, Martine Dunnwald1 1Department of Anatomy and Cell Biology, University of Iowa This protocol describes how to generate bilateral, full-thickness excisional wounds in mice and how to subsequently monitor, harvest, and prepare the wounds for morphometric analysis. Included is an in-depth description of how to use serial histological sections to define, precisely quantify and detect morphometric defects. Immunology and Infection Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions Kaitlin D Yarrington*1, Andrea Sánchez Peña*1, Dominique H Limoli1 1Department of Microbiology and Immunology, Carver College of Medicine, University of Iowa This live-bacterial cell imaging protocol allows for visualization of interactions between multiple bacterial species at the single-cell level over time. Time-lapse imaging allows for observation of each bacterial species in monoculture or coculture to interrogate interspecies interactions in multispecies bacterial communities, including individual cell motility and viability. Medicine A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development Brian A. Juber1, Timothy G. Elgin1, Erin M. Fricke2, Huyiu Gong1, Jeffrey Reese3, Steven J. McElroy1,4 1Division of Neonatology, Stead Family Department of Pediatrics, University of Iowa, 2Division of Maternal Fetal Medicine, Department of Obstetrics & Gynecology, University of Iowa, 3Division of Neonatology, Department of Pediatrics, Vanderbilt University, 4Department of Microbiology & Immunology, University of Iowa We developed a model of chorioamnionitis to simulate fetal exposure to maternal inflammation (FEMI) without complications of live organisms to examine the effects of FEMI on development of the offspring’s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis. Medicine Cooling or Warming the Esophagus to Reduce Esophageal Injury During Left Atrial Ablation in the Treatment of Atrial Fibrillation Jason Zagrodzky1, Mark M. Gallagher2, Lisa W. M. Leung2, Tiffany Sharkoski3, Pasquale Santangeli4, Cory Tschabrunn4, Jose M. Guerra5, Bieito Campos5, John MacGregor6, Jamal Hayat2, Brad Clark7, Alex Mazur8, Marcel Feher9, Martin Arnold9, Mark Metzl10, Jose Nazari10, Erik Kulstad11 1 The goal of this protocol is to describe the use of esophageal temperature modulation to counteract esophageal thermal injury from left atrial ablation for the treatment of atrial fibrillation. Environment Microalgae Cultivation and Biomass Quantification in a Bench-Scale Photobioreactor with Corrosive Flue Gases Hannah R. Molitor1, Deborah E. Williard1, Jerald L. Schnoor1 1Department of Civil and Environmental Engineering, University of Iowa Bench-scale, axenic cultivation facilitates microalgal characterization and productivity optimization before subsequent process scale-up. Photobioreactors provide the necessary control for reliable and reproducible microalgal experiments and can be adapted to safely cultivate microalgae with the corrosive gases (CO2, SO2, NO2) from municipal or industrial combustion emissions. Genetics Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens Robert Faris1, Mary M. Weber1 1Department of Microbiology and Immunology, University of Iowa Bacterial pathogens secrete proteins into the host that target crucial biological processes. Identifying the host pathways targeted by bacterial effector proteins is key to addressing molecular pathogenesis. Here, a method using a modified yeast suppressor and toxicity screen to elucidate host pathways targeted by toxic bacterial effector proteins is described. Biology Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing Shailesh K. Shahi1, Kasra Zarei2, Natalya V. Guseva1, Ashutosh K. Mangalam1,2,3,4 1Department of Pathology, University of Iowa, 2Medical Scientist Training Program, University of Iowa, 3Graduate Program in Immunology, University of Iowa, 4Graduate Program in Molecular Medicine, University of Iowa Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution. Genetics Lentiviral Mediated Gene Silencing in Human Pseudoislet Prepared in Low Attachment Plates Siming Liu1,2, Mikako Harata1,2, Joseph A. Promes1,2, Anthony J. Burand2,3, James A. Ankrum2,3, Yumi Imai1,2 1Department of Internal Medicine, Carver College of Medicine, University of Iowa, 2Fraternal Order of Eagles Diabetes Research Center, University of Iowa, 3Roy J. Carver Department of Biomedical Engineering, University of Iowa A protocol to create gene modified human pseudoislets from dispersed human islet cells that are transduced by lentivirus carrying short hairpin RNA (shRNA) is presented. This protocol utilizes readily available enzyme and culture vessels, can be performed easily, and produces genetically modified human pseudoislets suitable for functional and morphological studies. Medicine Combining Volumetric Capnography And Barometric Plethysmography To Measure The Lung Structure-function Relationship McKayla Seymour1, Elizabeth Pritchard1, Hassan Sajjad2, Erik P. Tomasson2, Cole M. Blodgett1, Harold Winnike4, Oana V. Paun3, Michael Eberlein2, Melissa L. Bates1,4 1Department of Health and Human Physiology, Department of Internal Medicine, University of Iowa, 2Pulmonary, Critical Care and Occupational Medicine Division, University of Iowa, 3Hematology, Oncology and Bone Marrow Transplant Division, University of Iowa, 4Institute for Clinical and Translational Science and Stead Family Department of Pediatrics, University of Iowa Here, we describe two measures of pulmonary function – barometric plethysmography, which allows the measurement of lung volume, and volumetric capnography, a tool to measure the anatomic dead space and airways uniformity. These techniques may be used independently or combined to assess airways function at different lung volumes. Genetics Informatic Analysis of Sequence Data from Batch Yeast 2-Hybrid Screens Venkatramanan Krishnamani1, Tabitha A. Peterson1, Robert C. Piper1, Mark A. Stamnes1 1Molecular Physiology and Biophysics, University of Iowa Deep sequencing of yeast populations selected for positive yeast 2-hybrid interactions potentially yields a wealth of information about interacting partner proteins. Here, we describe the operation of specific bioinformatics tools and customized updated software to analyze sequence data from such screens. Biochemistry A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions Tabitha A. Peterson1, Mark A. Stamnes1, Robert C. Piper1 1Molecular Physiology and Biophysics, University of Iowa Batch processing of yeast 2-hybrid screens allows for direct comparison of the interaction profiles of multiple bait proteins with a highly complex set of prey fusion proteins. Here, we describe refined methods, new reagents, and how to implement their use for such screens. Biochemistry Dissection of Human Retina and RPE-Choroid for Proteomic Analysis Thiago Cabral*1,2,7,8, Marcus A. Toral*3,4, Gabriel Velez3,4, James E. DiCarlo1,2, Anuradha M. Gore3, MaryAnn Mahajan3, Stephen H. Tsang1,2, Alexander G. Bassuk5,6, Vinit B. Mahajan3,9 1Barbara & Donald Jonas Stem Cell Laboratory, and Bernard & Shirlee Brown Glaucoma Laboratory, Department of Pathology & Cell Biology, Institute of Human Nutrition, College of Physicians and Surgeons, Columbia University, 2Edward S. Harkness Eye Institute, New York-Presbyterian Hospital, 3Omics Laboratory, Byers Eye Institute, Department of Ophthalmology, Stanford University, 4Medical Scientist Training Program, University of Iowa, 5Department of Pediatrics, University of Iowa, 6Department of Neurology, University of Iowa, 7Department of Ophthalmology, Federal University of Sao Paulo (UNIFESP), 8Department of Ophthalmology, Federal University of EspÍrito Santo (UFES), 9Palo Alto Veterans Administration, Palo Alto, CA The human retina is composed of functionally and molecularly distinct regions, including the fovea, macula, and peripheral retina. Here, we describe a method using punch biopsies and manual removal of tissue layers from a human eye to dissect and collect these distinct retinal regions for downstream proteomic analysis. Neuroscience In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin Arianna R. Lark1,2, Toshihiro Kitamoto2,3, Jean-René Martin1 1Equipe: Imagerie Cérébrale Fonctionnelle et Comportements (ICFC), Institut des Neurosciences Paris-Saclay (Nero-PSI), UMR-9197, CNRS/Université Paris Sud, 2Interdisciplinary Program in Neuroscience, Graduate College, University of Iowa, 3Department of Anesthesia, Carver College of Medicine, University of Iowa Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution. Neuroscience Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons Jin-Young Koh*1,2, Sadahiro Iwabuchi*1, Zhengmin Huang3, N. Charles Harata1 1Department of Molecular Physiology & Biophysics, University of Iowa Carver College of Medicine, 2Department of Psychiatry, University of Iowa Carver College of Medicine, 3EZ BioResearch LLC We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping. Biology Aip1p Dynamics Are Altered by the R256H Mutation in Actin Alyson R. Pierick1, Melissa McKane2, Kuo-Kuang Wen2, Heather L. Bartlett1,2 1Department of Pediatrics, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, 2Department of Biochemistry, Roy J. and Lucille A. Carver College of Medicine, University of Iowa Disease-causing mutations in actin can alter cytoskeletal function. Cytoskeletal dynamics are quantified through imaging of fluorescently tagged proteins using total internal fluorescence microscopy. As an example, the cytoskeletal protein, Aip1p, has altered localization and movement in cells expressing the mutant actin isoform, R256H. Bioengineering Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction Aribet M. De Jesús1, Edward A. Sander1 1Department of Biomedical Engineering, University of Iowa Time-lapse microscopy and image processing techniques were used to observe and analyze fibroblast-mediated gel compaction and fibrin fiber realignment in an environmentally controlled bioreactor over a 48 hr period. Biology The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation Andrew J. Spracklen1, Tina L. Tootle1 1Department of Anatomy and Cell Biology, University of Iowa Carver College of Medicine Stage-specific isolation of mid-to-late Drosophila follicles is useful for a variety of purposes. Such follicles develop in culture, which allows for genetic and/or pharmacologic manipulations to be coupled with in vitro development assays and live imaging. Additionally, follicles can be used for molecular studies, such as isolating mRNA and protein. Medicine Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye Katherine J. Wert1,2, Jessica M. Skeie3,4, Richard J. Davis1, Stephen H. Tsang1,3, Vinit B. Mahajan3,4 1Bernard and Shirlee Brown Glaucoma Laboratory, Department of Ophthalmology, Columbia University, 2Institute of Human Nutrition, College of Physicians & Surgeons, Columbia University, 3Omics Laboratory, University of Iowa, 4Department of Ophthalmology and Visual Sciences, University of Iowa This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye. Neuroscience Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones Andrew J. Shepherd1, Durga P. Mohapatra1,2 1Department of Pharmacology, The University of Iowa, 2Department of Anesthesia, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers. Medicine Mouse Eye Enucleation for Remote High-throughput Phenotyping Vinit B. Mahajan1,2, Jessica M. Skeie1,2, Amir H. Assefnia2,3, MaryAnn Mahajan1,2, Stephen H. Tsang2,4 1Department of Ophthalmology and Visual Sciences, University of Iowa, 2Omics Laboratory, University of Iowa, 3School of Dentistry, UCLA, 4Bernard and Shirlee Brown Glaucoma Laboratory, Department of Ophthalmology, College of Physicians and Surgeons, Columbia University The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens. Biology Evisceration of Mouse Vitreous and Retina for Proteomic Analyses Jessica M. Skeie1,2, Stephen H. Tsang3, Vinit B. Mahajan1,2 1Omics Laboratory, University of Iowa, 2Ophthalmology and Visual Sciences, University of Iowa, 3Harkness Eye Institute, Columbia University College of Physicians and Surgeons The dissection technique illustrates evisceration of the vitreous, retina, and lens from the mouse eye, separation by centrifugation, and characterization with protein assays. Neuroscience Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology Jeremy Duncan1, Jennifer Kersigo1, Brian Gray2, Bernd Fritzsch1 1Department of Biology, University of Iowa, 2Molecular Targeting Technologies, Inc. A combination of different techniques to maximize data collection from mouse tissue is presented. Medicine Dissection of Human Vitreous Body Elements for Proteomic Analysis Jessica M. Skeie1, Vinit B. Mahajan1 1Department of Ophthalmology and Visual Sciences, Omics Laboratory, University of Iowa This video shows an effective technique for differentiating and dissecting the various semi-transparent structures of the human vitreous body in post mortem eyes. Biology Fertilization of Xenopus oocytes using the Host Transfer Method Patricia N. Schneider1, Alissa M. Hulstrand1, Douglas W. Houston1 1Department of Biology, University of Iowa Procedure for fertilizing Xenopus oocytes by the host transfer method. Immunology and Infection In vivo Imaging of Transgenic Leishmania Parasites in a Live Host Colin J. Thalhofer1, Joel W. Graff2, Laurie Love-Homan3, Suzanne M. Hickerson4, Noah Craft5, Stephen M. Beverley4, Mary E. Wilson6,7 1Interdisciplinary Immunology Program, University of Iowa, and the VA Medical Center, 2Department of Biochemistry, University of Iowa, and the VA Medical Center, 3Department of Internal Medicine, University of Iowa, 4Department of Molecular Microbiology, Washington University School of Medicine, 5Division of Dermatology, Harbor-UCLA Medical Center, Hanley-Hardison Research Center, 6Interdisciplinary Immunology Program, Iowa City VA Medical Center, 7Departments of Internal Medicine, Microbiology and Epidemiology, University of Iowa An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.
