University of Cincinnati College of Medicine 25 articles published in JoVE Biology Guinea Pig Round Window Membrane Explantation for Ex Vivo Studies Sarek A. Shen1, Mukund Madhav Goyal2, Kelly Lane1, Mohamed Lehar1, Daniel Q. Sun1,3 1Department of Otolaryngology-Head & Neck Surgery, Johns Hopkins School of Medicine, 2Department of Chemical and Biomolecular Engineering, Johns Hopkins Whiting School of Engineering, 3Department of Otolaryngology-Head and Neck Surgery, University of Cincinnati College of Medicine This protocol outlines a method for the explantation of the round window membrane from guinea pig temporal bones, providing a valuable resource for ex vivo studies. Cancer Research Screening Ion Channels in Cancer Cells Laura Kallay1, Vaibhavkumar S. Gawali1, Donatien Kamdem Toukam1, Debanjan Bhattacharya1, Andrew Jenkins2, Soma Sengupta3, Daniel A. Pomeranz Krummel3 1Department of Neurology and Rehabilitation Medicine, Division of Neuro-Oncology, University of Cincinnati College of Medicine, 2Department of Pharmaceutical Sciences, School of Pharmacy, University of Saint Joseph, 3The Vontz Center for Molecular Studies, University of Cincinnati College of Medicine The pharmacological targeting of ion channels is a promising approach to treating solid tumors. Detailed protocols are provided for characterizing ion channel function in cancer cells and assaying the effects of ion channel modulators on cancer viability. Biology Quantitative Determination of De Novo Fatty Acid Synthesis in Brown Adipose Tissue Using Deuterium Oxide Rory Turner*1, Rajib Mukherjee*2, Martina Wallace1, Joan Sanchez-Gurmaches2,3,4 1UCD Conway Institute and UCD Institute of Food and Health, School of Agriculture and Food Science, University College Dublin, 2Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, 4Department of Pediatrics, University of Cincinnati College of Medicine Here, we present an inexpensive quantitative method utilizing deuterium oxide and gas chromatography mass spectrometry (GCMS) for the analysis of total fatty acid de novo lipogenesis in brown adipose tissue in vivo. Biology An Integrated Approach for Microprotein Identification and Sequence Analysis Omar Brito-Estrada*1, Keira R. Hassel*1, Catherine A. Makarewich1,2 1 The protocol described here provides detailed instructions on how to analyze genomic regions of interest for microprotein-coding potential using PhyloCSF on the user-friendly UCSC Genome Browser. Additionally, several tools and resources are recommended to further investigate sequence characteristics of identified microproteins to gain insight into their putative functions. Biology Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System Md Nasimuzzaman1,2, Sophia Villaveces1, Johannes C. M. van der Loo1,2,3, Sivani Alla1 1Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia In this protocol, AAV2 vector is produced by co-culturing Spodoptera frugiperda (Sf9) insect cells with baculovirus (BV)-AAV2-green fluorescent protein (GFP) or therapeutic gene and BV-AAV2-rep-cap infected Sf9 cells in suspension culture. AAV particles are released from the cells using detergent, clarified, purified by affinity column chromatography, and concentrated by tangential flow filtration. Developmental Biology A 3-D Tail Explant Culture to Study Vertebrate Segmentation in Zebrafish M. Fethullah Simsek1,3, Ertugrul M. Özbudak1,2,3 1 Here, we present the protocol for 3-D tissue culture of the zebrafish posterior body axis, enabling live study of vertebrate segmentation. This explant model provides control over axis elongation, alteration of morphogen sources, and subcellular resolution tissue-level live imaging. Behavior Using a Murine Model of Psychosocial Stress in Pregnancy as a Translationally Relevant Paradigm for Psychiatric Disorders in Mothers and Infants Sandra P. Zoubovsky1,2,3, Akil Wilder4, Louis Muglia1,2,3 1Division of Human Genetics, Cincinnati Children’s Hospital Medical Center, 3Department of Pediatrics, University of Cincinnati College of Medicine, 4 The chronic psychosocial stress (CGS) paradigm employs clinically relevant stressors during pregnancy in mice to model psychiatric disorders of mothers and infants. Here, we provide a step-by-step procedure of applying the CGS paradigm and downstream assessments to validate this model. Medicine Refined CLARITY-Based Tissue Clearing for Three-Dimensional Fibroblast Organization in Healthy and Injured Mouse Hearts Demetria M. Fischesser1,2, Evan C. Meyer3, Michelle Sargent2, Jeffery D. Molkentin2 1Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, 2 A refined method of tissue clearing was developed and applied to the adult mouse heart. This method was designed to clear dense, autofluorescent cardiac tissue, while maintaining labeled fibroblast fluorescence attributed to a genetic reporter strategy. Developmental Biology Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye Yu-Han Yeh*1,2, Mengwen Hu*1,2, Toshinori Nakagawa3,4, Akihiko Sakashita1,2, Shosei Yoshida3,4, So Maezawa5,6, Satoshi H. Namekawa1,2 1 Here we present a simple and efficient method to isolate live meiotic and post-meiotic germ cells from adult mouse testes. Using a low-cytotoxicity, violet-excited DNA binding dye and fluorescence-activated cell sorting, one can isolate highly enriched spermatogenic cell populations for many downstream applications. Developmental Biology A Patient-Derived Xenograft Model for Venous Malformation Sandra Schrenk*1, Jillian Goines*1, Elisa Boscolo1,2 1 We present a detailed protocol to generate a murine xenograft model of venous malformation. This model is based on the subcutaneous injection of patient-derived endothelial cells containing hyper-activating TIE2 and/or PIK3CA gene mutations. Xenograft lesions closely recapitulate the histopathological features of VM patient tissue. Bioengineering Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification Zhenyuan Xu1, Jacob A. Orkwis1, Greg M. Harris1,2,3 1Department of Chemical and Environmental Engineering, University of Cincinnati, 2Department of Biomedical Engineering, University of Cincinnati, 3Neuroscience Graduate Program, University of Cincinnati College of Medicine This methodology aims to illustrate the mechanisms by which extracellular matrix cues such as substrate stiffness, protein composition and cell morphology regulate Schwann cell (SC) phenotype. Immunology and Infection Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes Naoyoshi Sakitani*1, Takahiro Maekawa*1, Kumiko Saitou1,2, Katsuhiko Suzuki3, Shuhei Murase1,4, Masakuni Tokunaga1, Daisuke Yoshino5, Keisuke Sawada6, Atsushi Takashima7, Motoshi Nagao1, Toru Ogata1, Yasuhiro Sawada1,8 1Department of Rehabilitation for Motor Functions, National Rehabilitation Center for Persons with Disabilities, 2Graduate School of Sport Sciences, Waseda University, 3Faculty of Sport Sciences, Waseda University, 4Department of Orthopaedic Surgery, Graduate School of Medicine, The University of Tokyo, 5Frontier Research Institute for Interdisciplinary Sciences, Tohoku University, 6University of Cincinnati College of Medicine, 7Department of Assistive Technology, National Rehabilitation Center for Persons with Disabilities, 8Department of Clinical Research, National Rehabilitation Center for Persons with Disabilities Here we describe the protocols for applying defined mechanical loads to mouse calves and for monitoring the concomitant intramuscular pressure changes. The experimental systems that we have developed can be useful for investigating the mechanism behind the beneficial effects of physical exercise and massage. Immunology and Infection Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers Matthew J. Beucler1, William E. Miller1 1Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine We present a method for isolating and cultivating primary human salivary gland-derived epithelial cells. These cells exhibit gene expression patterns consistent with them being of salivary epithelial origin and can be grown as salispheres on basement membrane matrices derived from Engelbreth-Holm-Swarm tumor cells or as monolayers on treated culture dishes. Biology Purification of Platelets from Mouse Blood Marilou G. Narciso1, Md Nasimuzzaman1,2 1 Here, mouse blood was collected in the presence of an anti-coagulant. The platelets were purified by iohexol gradient medium using low speed centrifugation. The platelets were activated with thrombin to investigate if they were viable. The quality of the purified platelets was analyzed by flow cytometry and microscopy. Immunology and Infection Integrate Imaging Flow Cytometry and Transcriptomic Profiling to Evaluate Altered Endocytic CD1d Trafficking Manju Sharma1, Xiang Zhang1, Shouxiong Huang1 1Department of Environmental Health, University of Cincinnati College of Medicine Imaging flow cytometry provides an ideal approach to detect the morphological and functional alteration of cells at individual and populational levels. Disrupted endocytic function for lipid antigen presentation in pollutant-exposed human dendritic cells was demonstrated with a combined transcriptomic profiling of gene expression and morphological demonstration of protein trafficking. Genetics Production and Purification of Baculovirus for Gene Therapy Application Md Nasimuzzaman1,2, Johannes C.M. van der Loo1,2,3, Punam Malik1,2 1 In this protocol, baculovirus is produced by transient transfection of baculovirus plasmid into Sf9 cells and amplified in a serum-free suspension culture. The supernatant is purified by heparin affinity chromatography and further concentrated by ultracentrifugation. This protocol is useful for the production and purification of baculovirus for gene therapy application. Developmental Biology Studying Wnt Signaling During Patterning of Conducting Airways John Snowball1, Manoj Ambalavanan1, Debora Sinner1,2 1Neonatology and Pulmonary Biology-Perinatal Institute, Cincinnati Children’s Hospital Medical Center, 2University of Cincinnati, College of Medicine The use of reporter mice coupled to whole mount and section staining, microscopy and in vivo assays facilitates the analysis of mechanisms underlying the normal patterning of the respiratory tract. Here we describe how these techniques contributed to the analysis of Wnt signaling during tracheal development. Immunology and Infection Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis Nurit P. Azouz1,2, Mitsunori Fukuda3, Marc E. Rothenberg2, Ronit Sagi-Eisenberg1 1Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, 2Division of Allergy and Immunology, Department of Pediatrics, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, 3Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University The goal of the present protocol was to develop a method that will allow functional genomic analyses of mast cell secretion. The protocol is based on quantitative assessment of the release of a fluorescent reporter gene cotrasfected with the gene of interest and real time analyses of the secretory granule's morphology. Medicine Localization, Identification, and Excision of Murine Adipose Depots Adrien Mann1, Allie Thompson1, Nathan Robbins1, Andra L. Blomkalns1 1Department of Emergency Medicine, University of Cincinnati College of Medicine Due to the drastic and negative connection between obesity and other comorbidities, research on the role adipose plays in disease and overall health is warranted. We present a protocol for the isolation and excision of adipose depots allowing for the study of adipose using in situ and in vitro methods. Biology Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation Minghui Yue*1,2, John Lalith Charles Richard*1,2, Norishige Yamada1,2, Akiyo Ogawa1, Yuya Ogawa1,2 1 We developed an easily customized strand-specific fluorescent in situ hybridization (FISH) protocol combined with immunofluorescence. This allows for a detailed examination of RNA dynamics with simultaneous insight into the chromatin structure, nuclear organization, and transcriptional regulation at the single cell level. Medicine Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease Nathan Robbins1, Allie Thompson1, Adrien Mann1, Andra L. Blomkalns1 1Department of Emergency Medicine, University of Cincinnati College of Medicine Pathology of the aorta can lead to severe morbidity and mortality, therefore research of disease progression and potential therapies is warranted. Here, we present a protocol to isolate and excise the murine aorta to aid researchers in their investigation of cardiovascular disease. Medicine Implantation of Total Artificial Heart in Congenital Heart Disease Iki Adachi1,2, David S. L. Morales3 1 This is a case report of a patient with congenitally corrected transposition of the great arteries (CCTGA) who received a total artificial heart (TAH) as a bridge to heart transplant. The TAH was successfully implanted with modifications to accommodate the patient's congenitally malformed heart. Biology Identification of a Murine Erythroblast Subpopulation Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry Diamantis G. Konstantinidis1, Suvarnamala Pushkaran1, Katie Giger1, Stefanos Manganaris2, Yi Zheng1, Theodosia A. Kalfa1 1Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, 2IBM The present protocol describes a novel method of identifying a population of enucleating orthochromatic erythroblasts by multi-spectral imaging flow cytometry, providing a visualization of the erythroblast enucleation process. Medicine In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles Zhengtao Chu1,2, Kathleen LaSance3, Victor Blanco1, Chang-Hyuk Kwon5,6, Balveen Kaur5,6, Malinda Frederick4, Sherry Thornton4, Lisa Lemen3, Xiaoyang Qi1,2 1Division of Hematology-Oncology, Department of Internal Medicine, University of Cincinnati College of Medicine, 2Division of Human Genetics, University of Cincinnati College of Medicine, 3Department of Radiology, University of Cincinnati College of Medicine, 4Division of Rheumatology, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, 5Solid Tumor Biology Program, James Comprehensive Cancer Center, The Ohio State University Medical Center, 6Department of Neurological Surgery, James Comprehensive Cancer Center, The Ohio State University Medical Center We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes. Biology Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells Satoshi H. Namekawa1,2 1 The material here describes a method developed to preserve the three-dimensional chromatin structure of testicular germ cells. This has been termed the three-dimensional (3D) slide method. This method improves sensitivity for detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH).
