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University of Cincinnati College of Medicine

28 ARTICLES PUBLISHED IN JoVE

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Biology

Isolation and Primary Culture of Rat Hepatic Cells
Ling Shen 1, Allix Hillebrand 2, David Q.-H. Wang 3, Min Liu 1
1Department of Pathology and Laboratory Medicine, University of Cincinnati College of Medicine, 2American University in Washington, D.C., 3Department of Internal Medicine, Saint Louis University School of Medicine

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 × 108 cells per preparation with cell viability between 88 ~ 96%.

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Biology

Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies
Catherine Moorwood 1, Min Liu 2, Zuozhen Tian 2, Elisabeth R. Barton 3
1Department of Anatomy and Cell Biology, School of Dental Medicine, University of Pennsylvania, 2Department of Physiology, Perelman School of Medicine, University of Pennsylvania, 3Department of Anatomy and Cell Biology, School of Dental Medicine, School of Dental Medicine, University of Pennsylvania

Muscle function measurements contribute to the evaluation of potential therapeutics for muscle pathology, as well as to the determination of mechanisms underlying physiology of this tissue. We will demonstrate the preparation of the extensor digitorum longus and diaphragm muscles for functional testing. Protocols for isometric and eccentric contractions will be shown, as well as differences in results between dystrophic muscles, representing a pathological state, and wildtype muscles.

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Biology

Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells
Satoshi H. Namekawa 1,2
1Division of Reproductive Sciences, Division of Developmental Biology, Perinatal Institute, Cincinnati Children's Hospital Medical Center, 2Department of Pediatrics, University of Cincinnati College of Medicine

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).

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Biology

Identification of a Murine Erythroblast Subpopulation Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry
Diamantis G. Konstantinidis 1, Suvarnamala Pushkaran 1, Katie Giger 1, Stefanos Manganaris 2, Yi Zheng 1, Theodosia A. Kalfa 1
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.

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Medicine

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles
Zhengtao Chu 1,2, Kathleen LaSance 3, Victor Blanco 1, Chang-Hyuk Kwon 5,6, Balveen Kaur 5,6, Malinda Frederick 4, Sherry Thornton 4, Lisa Lemen 3, Xiaoyang Qi 1,2
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.

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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 Yamada 1,2, Akiyo Ogawa 1, Yuya Ogawa 1,2
1Division of Reproductive Sciences, Cincinnati Children's Hospital Medical Center, 2Department of Pediatrics, University of Cincinnati College of Medicine

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.

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Medicine

Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease
Nathan Robbins 1, Allie Thompson 1, Adrien Mann 1, Andra L. Blomkalns 1
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.

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Medicine

Localization, Identification, and Excision of Murine Adipose Depots
Adrien Mann 1, Allie Thompson 1, Nathan Robbins 1, Andra L. Blomkalns 1
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.

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Biochemistry

A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells
Xiongwei Cai 1,2,3, Yi Zheng 1, Nancy A. Speck 2
1Division of Experimental Hematology, Cincinnati Children's Hospital Medical Center, 2Department of Cell and Developmental Biology, Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, 3Department of Obstetrics and Gynecology, Southwest Hospital, Third Military Medical University

A standard Western blotting protocol was optimized for analyzing as few as 500 hematopoietic stem or progenitor cells. Optimization involves careful handling of the cell sample, limiting transfers between tubes, and directly lysing the cells in Laemmli sample buffer.

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Genetics

Production and Purification of Baculovirus for Gene Therapy Application
Md Nasimuzzaman 1,2, Johannes C.M. van der Loo 1,2,3, Punam Malik 1,2
1Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, 2University of Cincinnati College of Medicine, 3Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia

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.

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Immunology and Infection

Integrate Imaging Flow Cytometry and Transcriptomic Profiling to Evaluate Altered Endocytic CD1d Trafficking
Manju Sharma 1, Xiang Zhang 1, Shouxiong Huang 1
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.

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Medicine

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer
Ligia B. Schmitd 1, Min Liu 1, Christina S. Scanlon 1, Rajat Banerjee 1, Nisha J. D’Silva 1,2
1Periodontics and Oral Medicine, University of Michigan School of Dentistry, 2Pathology, University of Michigan Medical School

Perineural invasion is an aggressive phenotype for head and neck squamous cell carcinomas and other tumors. The chick chorioallantoic membrane model has been used for studying angiogenesis, cancer invasion, and metastasis. Here we demonstrate how this model can be utilized to assess perineural invasion in vivo.

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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 Saitou 1,2, Katsuhiko Suzuki 3, Shuhei Murase 1,4, Masakuni Tokunaga 1, Daisuke Yoshino 5, Keisuke Sawada 6, Atsushi Takashima 7, Motoshi Nagao 1, Toru Ogata 1, Yasuhiro Sawada 1,8
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.

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Biology

Purification of Platelets from Mouse Blood
Marilou G. Narciso 1, Md Nasimuzzaman 1,2
1Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, 2University of Cincinnati College of Medicine

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.

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JoVE Core

Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers
Matthew J. Beucler 1, William E. Miller 1
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.

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Engineering

Graphene-Assisted Quasi-van der Waals Epitaxy of AlN Film on Nano-Patterned Sapphire Substrate for Ultraviolet Light Emitting Diodes
Xiang Zhang *1,2, Zhaolong Chen *3, Hongliang Chang 1,2, Jianchang Yan 1,2, Shenyuan Yang 2,4, Junxi Wang 1,2, Peng Gao 5, Tongbo Wei 1,2
1State Key Laboratory of Solid-State Lighting, Institute of Semiconductors, Chinese Academy of Sciences, 2Center of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Science, 3Center for Nanochemistry (CNC), Beijing National Laboratory for Molecular Science, College of Chemistry and Molecular Engineering, Peking University, 4State Key Laboratory of Superlattices and Microstructures, Institute of Semiconductors, Chinese Academy of Sciences, 5Electron Microscopy Laboratory, School of Physics, Peking University

A protocol for graphene-assisted growth of high-quality AlN films on nano-patterned sapphire substrate is presented.

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Bioengineering

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification
Zhenyuan Xu 1, Jacob A. Orkwis 1, Greg M. Harris 1,2,3
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.

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Developmental Biology

A Patient-Derived Xenograft Model for Venous Malformation
Sandra Schrenk *1, Jillian Goines *1, Elisa Boscolo 1,2
1Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, 2Department of Pediatrics, University of Cincinnati College of Medicine

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.

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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 Nakagawa 3,4, Akihiko Sakashita 1,2, Shosei Yoshida 3,4, So Maezawa 5,6, Satoshi H. Namekawa 1,2
1Division of Reproductive Sciences, Division of Developmental Biology, Perinatal Institute, Cincinnati Children's Hospital Medical Center, 2Department of Pediatrics, University of Cincinnati College of Medicine, 3Division of Germ Cell Biology, National Institute for Basic Biology, National Institutes of Natural Sciences, 4Department of Basic Biology, School of Life Science, Graduate University for Advanced Studies (Sokendai), 5Department of Animal Science and Biotechnology, School of Veterinary Medicine, Azabu University, 6Faculty of Science and Technology, Department of Applied Biological Science, Tokyo University of Science

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.

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Developmental Biology

A 3-D Tail Explant Culture to Study Vertebrate Segmentation in Zebrafish
M. Fethullah Simsek 1,3, Ertugrul M. Özbudak 1,2,3
1Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, 2Department of Pediatrics, University of Cincinnati College of Medicine, 3Department of Genetics, Albert Einstein College of Medicine

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.

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Medicine

Refined CLARITY-Based Tissue Clearing for Three-Dimensional Fibroblast Organization in Healthy and Injured Mouse Hearts
Demetria M. Fischesser 1,2, Evan C. Meyer 3, Michelle Sargent 2, Jeffery D. Molkentin 2
1Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, 2Division of Molecular Cardiovascular Biology, Cincinnati Children's Hospital Medical Center, 3Confocal Imaging Core, Cincinnati Children's Hospital Medical Center

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.

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Behavior

Using a Murine Model of Psychosocial Stress in Pregnancy as a Translationally Relevant Paradigm for Psychiatric Disorders in Mothers and Infants
Sandra P. Zoubovsky 1,2,3, Akil Wilder 4, Louis Muglia 1,2,3
1Molecular Developmental Biology Program, Cincinnati Children’s Hospital Medical Center, 2Division of Human Genetics, Cincinnati Children’s Hospital Medical Center, 3Department of Pediatrics, University of Cincinnati College of Medicine, 4Division of Veterinary Services, Cincinnati Children’s Hospital Medical Center

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.

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Biology

Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System
Md Nasimuzzaman 1,2, Sophia Villaveces 1, Johannes C. M. van der Loo 1,2,3, Sivani Alla 1
1Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, 2University of Cincinnati College of Medicine, 3Raymond 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.

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Biology

An Integrated Approach for Microprotein Identification and Sequence Analysis
Omar Brito-Estrada *1, Keira R. Hassel *1, Catherine A. Makarewich 1,2
1The Heart Institute, Division of Molecular Cardiovascular Biology, Cincinnati Children's Hospital Medical Center, 2Department of Pediatrics, University of Cincinnati College of Medicine

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.

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Biology

Quantitative Determination of De Novo Fatty Acid Synthesis in Brown Adipose Tissue Using Deuterium Oxide
Rory Turner *1, Rajib Mukherjee *2, Martina Wallace 1, Joan Sanchez-Gurmaches 2,3,4
1UCD Conway Institute and UCD Institute of Food and Health, School of Agriculture and Food Science, University College Dublin, 2Division of Endocrinology, Cincinnati Children’s Hospital Medical Center, Cincinnati, 3Division 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.

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Cancer Research

Screening Ion Channels in Cancer Cells
Laura Kallay 1, Vaibhavkumar S. Gawali 1, Donatien Kamdem Toukam 1, Debanjan Bhattacharya 1, Andrew Jenkins 2, Soma Sengupta 3, Daniel A. Pomeranz Krummel 3
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.

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Biology

Guinea Pig Round Window Membrane Explantation for Ex Vivo Studies
Sarek A. Shen 1, Mukund Madhav Goyal 2, Kelly Lane 1, Mohamed Lehar 1, Daniel Q. Sun 1,3
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.

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Biology

Monitoring Circadian Oscillations with a Bioluminescence Reporter in Organoids
Sevde Goker 1, Suengwon Lee 1, Christian I. Hong 1
1Department of Pharmacology and Systems Physiology, University of Cincinnati College of Medicine

Circadian rhythms, which exist in most organisms, regulate the temporal organization of biological processes. 3D organoids have recently emerged as a physiologically relevant in vitro model. This protocol describes the use of bioluminescent reporters to observe circadian rhythms in organoids, enabling in vitro investigations of circadian rhythms in multicellular systems.

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