S'identifier

Johns Hopkins University School of Medicine

113 ARTICLES PUBLISHED IN JoVE

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Biology

ALS - Motor Neuron Disease: Mechanism and Development of New Therapies
Jeffrey D. Rothstein 1
1Department of Neurology, Johns Hopkins University

Jeffrey D. Rothstein speaks about the pathology and mechanisms underlying amyotrophic lateral sclerosis or ALS, advances in ALS research, and current strategies towards the development of therapies.

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Biology

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior
Bong Geun Chung 1, Amir Manbachi 1, Ali Khademhosseini 1
1Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology; Center for Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

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Biology

A Gradient-generating Microfluidic Device for Cell Biology
Bong Geun Chung 1, Amir Manbachi 1, Wajeeh Saadi 1, Francis Lin 1, Noo Li Jeon 1, Ali Khademhosseini 1
1Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology; Center for Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

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Immunology and Infection

Orthotopic Hind-Limb Transplantation in Rats
Robert Sucher *1, Rupert Oberhuber *1, Christian Margreiter 1, Guido Rumberg 1, Rishi Jindal 2, WP Andrew Lee 2, Raimund Margreiter 1, Johann Pratschke 1, Stefan Schneeberger 1, Gerald Brandacher 1
1Department of Visceral, Transplant, and Thoracic Surgery, Daniel Swarovski Research Laboratory, Innsbruck Medical University, 2Department of Surgery, Division of Plastic and Reconstructive Surgery, University of Pittsburgh Medical Center

Here we describe the orthotopic rat hind-limb transplantation procedure, which seems to be the gold standard in vivo model for composite tissue allotransplantation research.

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Neuroscience

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5
Tarran J. Pierfelice 1, Nicholas Gaiano 1,2
1Institute for Cell Engineering Neuroregeneration and Stem Cell Programs, Johns Hopkins University School of Medicine, 2Departments of Neurology, Neuroscience, and Oncology, Johns Hopkins University School of Medicine

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.

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Biology

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts
Zeshaan Rasheed 1, Qiuju Wang 1, William Matsui 1
1Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

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Neuroscience

In vivo Electroporation of Developing Mouse Retina
Jimmy de Melo 1, Seth Blackshaw 1,2,3,4,5
1Solomon H. Snyder Department of Neuroscience, Johns Hopkins School of Medicine, 2Department of Neurology, Johns Hopkins School of Medicine, 3Department of Ophthalmology, Johns Hopkins School of Medicine, 4Center for High-Throughput Biology, Johns Hopkins School of Medicine, 5Institute for Cell Engineering, Johns Hopkins School of Medicine

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

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Bioengineering

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal Skin and Melanoma Progression
Ling Li 1, Mizuho Fukunaga-Kalabis 1, Meenhard Herlyn 1
1Molecular and Cellular Oncogenesis Program, The Wistar Institute

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

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Biology

Drosophila Pupal Abdomen Immunohistochemistry
Wei Wang 1, John H. Yoder 1
1Department of Biological Sciences, University of Alabama

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

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Neuroscience

Derivation of Glial Restricted Precursors from E13 mice
André W. Phillips 1,2, Sina Falahati 1,2, Roshi DeSilva 1,3, Irina Shats 2, Joel Marx 1, Edwin Arauz 1, Douglas A. Kerr 4, Jeffrey D. Rothstein 2,5, Michael V. Johnston 1,2,6, Ali Fatemi 1,2,6
1Hugo W. Moser Research Institute at Kennedy Krieger, Johns Hopkins University, 2Department of Neurology, Johns Hopkins School of Medicine, 3University of Maryland , 4Experimental Neurology, Biogen Idec, 5The Brain Science Institute, Johns Hopkins School of Medicine, 6Department of Pediatrics, Johns Hopkins School of Medicine

This protocol outlines the derivation of Glial Restricted Precursors from fetal spinal cords and maintained in vitro either for transplantation or for the study of oligodendrocytic lineage.

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Medicine

Immunohistochemical Staining of B7-H1 (PD-L1) on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue
Elaine Bigelow 1,2, Katherine M. Bever 1,2, Haiying Xu 1,2,3, Allison Yager 1, Annie Wu 1,2,4, Janis Taube 3,5, Lieping Chen 6, Elizabeth M. Jaffee 1,2,5,7,8, Robert A. Anders 1,2,5,8, Lei Zheng 1,2,4,5,7
1The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, 2Department of Oncology, The Johns Hopkins University School of Medicine, 3Department of Dermatology, The Johns Hopkins University School of Medicine, 4Department of Surgery, Johns Hopkins University School of Medicine, 5The Sol Goldman Pancreatic Cancer Center, The Johns Hopkins University School of Medicine, 6Yale Cancer Center, Yale School of Medicine, 7The Skip Viragh Center for Pancreatic Cancer, The Johns Hopkins University School of Medicine, 8Department of Pathology, The Johns Hopkins University School of Medicine

B7-H1 (PD-L1) and its binding to PD-1 provide a major tumor-induced immunosuppressive signal in the tumor’s microenvironment. An immunohistochemical staining technique to characterize the expression and localization of B7-H1 in pancreatic adenocarcinoma is described here.

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Neuroscience

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons
Saijilafu 1, Feng-Quan Zhou 1,2
1Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, 2Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

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Medicine

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window
Lifeng Zhang *1, Andree Lapierre *1, Brittany Roy 1, Maili Lim 1, Jennifer Zhu 1, Wei Wang 1, Stephen B. Sampson 1, Kyuson Yun 1, Bonnie Lyons 1, Yun Li 1, Da-Ting Lin 1
1The Jackson Laboratory

By combining a polished and reinforced thin-skull (PoRTS) cranial window and glioblastoma (GBM) cell injection, we can observe glioma initiation and growth from injected GBM cells in the brain of a live mouse longitudinally.

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Neuroscience

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons
Yun Li 1, Brittany D. Roy 1, Wei Wang 1, Lifeng Zhang 1, Stephen B. Sampson 1, Da-Ting Lin 1
1The Jackson Laboratory

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

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Biology

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)
Zach Hensel 1, Xiaona Fang 1,2,3, Jie Xiao 1
1Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, 2Changchun Institute of Applied Chemistry, Chinese Academy of Sciences , 3Department of Physics, Jilin University

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the λ repressor CI 1.

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Biology

Super-resolution Imaging of the Bacterial Division Machinery
Jackson Buss 1, Carla Coltharp 1, Jie Xiao 1
1Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine

We describe a super-resolution imaging method to probe the structural organization of the bacterial FtsZ-ring, an essential apparatus for cell division. This method is based on quantitative analyses of photoactivated localization microscopy (PALM) images and can be applied to other bacterial cytoskeletal proteins.

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Bioengineering

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry
Ron B. Shmueli 1,2, Nupura S. Bhise 1,2, Jordan J. Green 1,2,3,4
1Biomedical Engineering Department, Johns Hopkins University School of Medicine, 2Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, 3Wilmer Eye Institute, Johns Hopkins University School of Medicine, 4Institute for Nanobiotechnology, Johns Hopkins University School of Medicine

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

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Medicine

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation (VCA) Research
Zuhaib Ibrahim 1, Damon S. Cooney 1, Jaimie T. Shores 1, Justin M. Sacks 1, Eric G. Wimmers 1, Steven C. Bonawitz 1, Chad Gordon 1, Dawn Ruben 1, Stefan Schneeberger 1, W. P. Andrew Lee 1, Gerald Brandacher 1
1Plastic and Reconstructive Surgery, Johns Hopkins University School of Medicine

Vascularized Composite Allotransplantations (VCA) have become a clinical reality. However, broad clinical application of VCA is limited by chronic multi-drug immunosuppression. The authors present a reliable and reproducible large animal model to translate novel immunomodulatory strategies that can minimize or potentially eliminate the need of immunosuppression in VCA.

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Immunology and Infection

Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells
Qiao Li 1, Lin Lu 1, Huimin Tao 1, Carolyn Xue 1, Seagal Teitz-Tennenbaum 2, John H. Owen 3, Jeffrey S Moyer 3, Mark E.P. Prince 3, Alfred E. Chang 1, Max S. Wicha 2
1Department of Surgery, University of Michigan, 2Department of Internal Medicine, University of Michigan, 3Department of Otolaryngology, University of Michigan

Evaluated in syngeneic immunocompetent hosts, cancer stem cell (CSC) based dendritic cell (DC) vaccine demonstrated significantly higher antitumor immunity than traditional DC vaccines pulsed with heterogeneous bulk tumor cells.

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Neuroscience

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition
Daniel A. Lee 1,2, Juan Salvatierra 2, Esteban Velarde 3, John Wong 3, Eric C. Ford 4, Seth Blackshaw 2,5
1Division of Biology, California Institute of Technology, 2Solomon H. Snyder Department of Neuroscience, Neurology, and Ophthalamology, Johns Hopkins University School of Medicine, 3Department of Radiation Oncology & Molecular Radiation Sciences, Johns Hopkins University School of Medicine, 4Department of Radiation Oncology, University Of Washington Medical Center, 5Institute for Cell Engineering and High-Throughput Biology Center, Johns Hopkins University School of Medicine

The function of adult-born mammalian neurons remains an active area of investigation. Ionizing radiation inhibits the birth of new neurons. Using computer tomography-guided focal irradiation (CFIR), three-dimensional anatomical targeting of specific neural progenitor populations can now be used to assess the functional role of adult neurogenesis.

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Medicine

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique
Rupert Oberhuber *1, Benno Cardini *1, Markus Kofler 1, Paul Ritschl 1, Robert Oellinger 1, Felix Aigner 1, Robert Sucher 1, Stefan Schneeberger 1, Johann Pratschke 1, Gerald Brandacher 2, Manuel Maglione 1
1Center of Operative Medicine, Department of Visceral, Transplant and Thoracic Surgery, Innsbruck Medical University, 2Department of Plastic and Reconstructive Surgery, Johns Hopkins University School of Medicine

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.

Herein, we provide additional details of this technique to supplement the video.

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Bioengineering

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides
Yang Li 1, Catherine A. Foss 2, Martin G. Pomper 2,3, S. Michael Yu 1,3
1Department of Bioengineering, University of Utah, 2Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, 3Institute for NanoBiotechnology, Johns Hopkins University

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

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Immunology and Infection

Isolation of Double Negative αβ T Cells from the Kidney
Maria N. Martina 1, Samatha Bandapalle 2, Hamid Rabb 2, Abdel R. Hamad 1
1Department of Pathology, Johns Hopkins University School of Medicine, 2Department of Medicine, Johns Hopkins University School of Medicine

DNabT cells are rare among peripheral T cells; however, they are abundant in certain non-lymphoid tissues. Difficulty of isolating DN T cells from non-lymphoid tissue hinders their functional analysis despite increasing recognized pathophysiologic significance. We describe a novel protocol for isolation of highly purified DN T cells from murine kidney.

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Biology

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus
In Young Choi 1, HoTae Lim 1, Gabsang Lee 1
1Institute for Cell Engineering, Department of Neurology and Neuroscience, Johns Hopkins University School of Medicine

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

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Biology

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR
HoTae Lim 1, In Young Choi 1, Gabsang Lee 1
1Institute for Cell Engineering, Department of Neurology and Neuroscience, Johns Hopkins University School of Medicine

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

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Medicine

Combined In vivo Optical and µCT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice
Nicholas M. Bernthal 1, Brad N. Taylor 2, Jeffrey A. Meganck 2, Yu Wang 3, Jonathan H. Shahbazian 3, Jared A. Niska 1, Kevin P. Francis 2, Lloyd S. Miller 3,4
1Orthopaedic Hospital Research Center, Orthopaedic Hospital Department of Orthopaedic Surgery, David Geffen School of Medicine at University of California, Los Angeles (UCLA), 2PerkinElmer, 3Department of Dermatology, Johns Hopkins University School of Medicine, 4Department of Medicine, Division of Infectious Diseases, Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine

Combined optical and μCT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

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Medicine

A Preclinical Murine Model of Hepatic Metastases
Kevin C. Soares 1, Kelly Foley 2, Kelly Olino 1, Ashley Leubner 2, Skye C. Mayo 1, Ajay Jain 1, Elizabeth Jaffee 2, Richard D. Schulick 3, Kiyoshi Yoshimura 1,2, Barish Edil 1,2, Lei Zheng 1,2
1Department of Surgery, The Johns Hopkins University School of Medicine, 2Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, 3Department of Surgery, University of Colorado Anschutz Medical Campus

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

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Biology

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis
Ho Lam Tang 1,2,3, Ho Man Tang 1,2,3, J. Marie Hardwick 1, Ming Chiu Fung 2
1W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, 2School of Life Sciences, Chinese University of Hong Kong, 3Center for Cell Dynamics, Department of Biological Chemistry, Johns Hopkins University School of Medicine

The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.

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Biology

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry
Subarna Bhattacharya *1, Paul W. Burridge *2, Erin M. Kropp 1, Sandra L. Chuppa 1, Wai-Meng Kwok 3, Joseph C. Wu 2, Kenneth R. Boheler 4,5, Rebekah L. Gundry 1,6
1Department of Biochemistry, Medical College of Wisconsin, 2Stanford Cardiovascular Institute, Stanford University School of Medicine, 3Department of Anesthesiology, Medical College of Wisconsin, 4Stem Cell and Regenerative Medicine Consortium, LKS Faculty of Medicine, Hong Kong University, 5Division of Cardiology, Johns Hopkins University School of Medicine, 6Cardiovascular Research Center, Biotechnology and Bioengineering Center, Medical College of Wisconsin

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

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Biology

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity
Nathachit Limjunyawong 1, Jonathan Fallica 1, Amritha Ramakrishnan 2, Kausik Datta 3, Matthew Gabrielson 1, Maureen Horton 1, Wayne Mitzner 1
1Department of Environmental Health Sciences, Johns Hopkins University Bloomberg School of Public Health, 2Department of International Health, Johns Hopkins University Bloomberg School of Public Health, 3Department of Medicine, Johns Hopkins University School of Medicine

We describe a means to quickly and simply measure the lung diffusing capacity in mice and show that it is sufficiently sensitive to phenotype changes in multiple common lung pathologies. This metric thus brings direct translational relevance to the mouse models, since diffusing capacity is also easily measured in humans.

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Developmental Biology

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche
Peter Andersen 1, Chulan Kwon 1
1Division of Cardiology, Institute for Cell Engineering, Johns Hopkins University School of Medicine

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

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Chemistry

Facile and Efficient Preparation of Tri-component Fluorescent Glycopolymers via RAFT-controlled Polymerization
Wei Wang 1, John M. Lester 1, Anthony E. Amorosa 2, Deborah L. Chance 3, Valeri V. Mossine 1, Thomas P. Mawhinney 1,4
1Department of Biochemistry, University of Missouri, 2Department of Biological Sciences, University of Missouri, 3Department of Molecular Microbiology & Immunology, University of Missouri, 4Department of Child Health, University of Missouri

An efficient, three-step synthesis of RAFT-based fluorescent glycopolymers, consisting of glycomonomer preparation, copolymerization, and post-modification, is demonstrated. This protocol can be used to prepare RAFT-based statistical glycopolymers with desired structures.

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Bioengineering

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications
Xiomara Calderón-Colón 1, Giorgio Raimondi 2, Jason J. Benkoski 1, Julia B. Patrone 3
1Research and Exploratory Development Department, The Johns Hopkins Applied Physics Laboratory, 2Department of Plastic and Reconstructive Surgery, Johns Hopkins School of Medicine, 3Asymmetric Operations Sector, The Johns Hopkins Applied Physics Laboratory

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

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Medicine

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice
Byoungchol Oh *1, Georg J. Furtmüller *1, Michael Sosin *1, Madeline L. Fryer 1, Lawrence J. Gottlieb 2,3, Michael R. Christy 4, Gerald Brandacher 1,5,6, Amir H. Dorafshar 1,5,6
1Johns Hopkins University School of Medicine, 2Burn and Complex Wound Center, 3Section of Plastic and Reconstructive Surgery, University of Chicago Medical Center, 4Division of Plastic, Reconstructive, and Maxillofacial Surgery, R Adams Cowley Shock Trauma Center, 5Department of Plastic and Reconstructive Surgery, Johns Hopkins University School of Medicine, 6Vascularized Composite Allotransplantation (VCA) Lab, Johns Hopkins University School of Medicine

To study combined solid organ and vascularized composite allotransplantation, we describe a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique.

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Medicine

Adapted Resistance Training Improves Strength in Eight Weeks in Individuals with Multiple Sclerosis
Jennifer L. Keller 1, Nora Fritz 1,2, Chen Chun Chiang 1, Allen Jiang 1, Tziporah Thompson 3, Nicole Cornet 1, Scott D. Newsome 4, Peter A. Calabresi 4, Kathleen Zackowski 1,2,4
1Motion Analysis Laboratory, Kennedy Krieger Institute, 2Physical Medicine & Rehabilitation, Johns Hopkins University School of Medicine, 3Johns Hopkins University School of Medicine, 4Department of Neurology, Johns Hopkins University School of Medicine

Hip weakness is a common symptom affecting walking ability in people with multiple sclerosis. Isolated muscle strengthening is a useful method to target specific weaknesses. This protocol describes a progressive resistance-training program using exercise bands to increase hip muscle strength.

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Medicine

Orthotopic Hind Limb Transplantation in the Mouse
Georg J. Furtmüller *1, Byoungchol Oh *1, Johanna Grahammer *2, Cheng-Hung Lin 3, Robert Sucher 4, Madeline L. Fryer 1, Giorgio Raimondi 1, W.P. Andrew Lee 1, Gerald Brandacher 1
1Department of Plastic and Reconstructive Surgery, Vascularized Composite Allotransplantation (VCA) Laboratory, Johns Hopkins University School of Medicine, 2Department of Visceral, Transplant and Thoracic Surgery, Innsbruck Medical University, 3Center for Vascularized Composite Allotransplantation, Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital and School of Medicine, 4Department of General, Visceral and Transplant Surgery, Charite Berlin

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

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Developmental Biology

Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning
Jason T. Schott *1, Leslie A. Kirby *1, Peter A. Calabresi 1, Emily G. Baxi 1
1Neurology, Johns Hopkins University, School of Medicine

Oligodendrocytes are the myelinating cells of the central nervous system. This protocol describes a method for the isolation and culture of their precursors, oligodendrocyte progenitor cells, from rat cortices, as well as a fast and reliable quantitative method to evaluate oligodendrogenesis in vitro in response to experimental factors.

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Immunology and Infection

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation
Sarah R. Amend 1, Kenneth C. Valkenburg 1, Kenneth J. Pienta 1
1Department of Urology, Johns Hopkins University

Here we present a protocol for the dissection of hind limb long bones (femurs and tibiae) from the laboratory mouse. We further describe a rapid technique for bone marrow isolation from these bones that utilizes centrifugation for removal of bone marrow from the bone marrow space.

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Medicine

Murine Prostate Micro-dissection and Surgical Castration
Kenneth C. Valkenburg 1, Sarah R. Amend 1, Kenneth J. Pienta 1
1Department of Urology, Johns Hopkins University

This manuscript describes the protocols for prostate micro-dissection and surgical castration in the laboratory mouse. We also depict representative results produced by these protocols. Finally, we discuss the advantages and utilization of these protocols.

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Biology

In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila
Ho Lam Tang 1, Ho Man Tang 1, Ming Chiu Fung 2, J. Marie Hardwick 1
1W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, 2School of Life Sciences, Chinese University of Hong Kong

To detect healthy cells in whole animals that contain low levels of caspase activity, the highly sensitive biosensor designated CaspaseTracker was generated for Drosophila. Caspase-dependent biosensor activity is detected in long-lived healthy cells throughout the internal organs of adult animals reared under optimized conditions in the absence of death stimuli.

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Medicine

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile
Ling Li 1, Klaus B. Piontek 1, Vivek Kumbhari 1, Masaharu Ishida 2, Florin M. Selaru 1
1Division of Gastroenterology and Hepatology, Department of Medicine, Johns Hopkins University School of Medicine, 2Department of Surgery, Tohoku University

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

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Medicine

Detecting Anastasis In Vivo by CaspaseTracker Biosensor
Ho Man Tang 1,2, Ming Chiu Fung 2, Ho Lam Tang 3
1Institute for Basic Biomedical Sciences, Johns Hopkins University School of Medicine, 2School of Life Sciences, Chinese University of Hong Kong, 3Department of Neurosurgery, Johns Hopkins University School of Medicine

Anastasis is technically challenging to detect in vivo because the cells that have reversed the cell death process can be morphologically indistinguishable from normal healthy cells. Here we describe protocols for detecting and tracking cells that undergo anastasis in live animals by using our newly developed in vivo CaspaseTracker biosensor system.

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JoVE Core

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo
Mikko Juusola 1,2, An Dau 2, Lei Zheng 1, Diana Rien 1,2
1National Key Laboratory of Cognitive Neuroscience and Learning, Beijing Normal University, 2Department of Biomedical Science, The University of Sheffield

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

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Neuroscience

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer
Chun-Chieh Lin 1, Olena Riabinina 2, Christopher J. Potter 1
1The Solomon H. Snyder Department of Neuroscience, Center for Sensory Biology, Johns Hopkins University School of Medicine, 2MRC Clinical Sciences Center, Imperial College London

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.

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Neuroscience

Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue
F. Javier Rubio *1, Xuan Li *1, Qing-Rong Liu 1, Raffaello Cimbro 2, Bruce T. Hope 1
1Behavioral Neuroscience Research Branch, Intramural Research Program, NIDA, NIH, DHHS, 2Division of Rheumatology, School of Medicine, Johns Hopkins University

Here we present a Fluorescence Activated Cell Sorting (FACS) protocol to study molecular alterations in Fos-expressing neuronal ensembles from both fresh and frozen brain tissue. The use of frozen tissue allows FACS isolation of many brain areas over multiple sessions to maximize the use of valuable animal subjects.

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Biochemistry

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling
Sarah A. Head 1, Jun O. Liu 1,2
1Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, 2Department of Oncology, Johns Hopkins University School of Medicine

We describe here a method for identification of small molecule-binding proteins using photoaffinity labeling. The advantage of this technique is that binding and covalent labeling of the target proteins occurs within the live cellular environment, removing the risk of disrupting native protein structure and binding conditions upon cell lysis.

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Medicine

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique
Benno Cardini *1, Rupert Oberhuber *1, Sven R Hein 1, Rebecca Eiter 1, Martin Hermann 2, Markus Kofler 1,3, Stefan Schneeberger 1, Gerald Brandacher 1,4, Manuel Maglione 1
1Center of Operative Medicine, Department of Visceral, Transplant and Thoracic Surgery, Medical University Innsbruck, 2Department of Anesthesiology and Critical Care Medicine, Medical University Innsbruck, 3Department of Cardiac Surgery, Medical University Innsbruck, 4Department of Plastic and Reconstructive Surgery, Vascularized Composite Allotransplantation (VCA) Laboratory, Johns Hopkins University School of Medicine

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

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Immunology and Infection

Murine Full-thickness Skin Transplantation
Chih-Hsien Cheng *1,2, Chen-Fang Lee *1,2, Madeline Fryer *3, Georg J. Furtmüller 3, Byoungchol Oh 3, Jonathan D. Powell 1, Gerald Brandacher 3
1Sidney-Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, 2Department of Liver and Transplantation Surgery, Chang-Gung Transplantation Institute, Chang-Gung Memorial Hospital, Chang-Gung University College of Medicine, 3Vascularized Composite Allotransplantation Laboratory, Department of Plastic and Reconstructive Surgery, Johns Hopkins University School of Medicine

Murine full-thickness skin transplantation is a well-established model to study rejection in an alloimmune setting. Here, we provide a tutorial of each step involved in performing a BALB/c-->C57BL/6 full-thickness skin transplant.

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Developmental Biology

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells
Yu Wang 1, Sharmin Alhaque 1, Kate Cameron 1, Jose Meseguer-Ripolles 1, Baltasar Lucendo-Villarin 1, Hassan Rashidi 1, David C. Hay 1
1MRC Centre for Regenerative Medicine, University of Edinburgh

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

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Education

Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding
Chirag Vasavda *1, Nicholas W. Zaccor *1, Paul C. Scherer 1, Charlotte J. Sumner 1,2, Solomon H. Snyder 1,3,4
1The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, 2Department of Neurology and neurosurgery, Johns Hopkins University School of Medicine, 3Departments of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, 4Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine

Guanosine triphosphate (GTP) binding is one of the earliest events in G-Protein-Coupled Receptor (GPCR) activation. This protocol describes how to pharmacologically characterize specific GPCR-ligand interactions by monitoring the binding of the radio-labeled GTP analog, [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTPγS), in response to a ligand of interest.

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Neuroscience

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila
Mingkuan Sun 1, Liam L. Chen 1
1Division of Neuropathology, Department of Pathology, Johns Hopkins University School of Medicine

Here, we describe a new approach to inflict closed-head traumatic brain injury in Drosophila melanogaster. Our method has the advantage of directly delivering repetitive impacts with adjustable strength to the head alone. Further exploration of the invertebrate system will help to illuminate the pathogenesis of chronic traumatic encephalopathy.

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JoVE Journal

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy
Lijuan He *1,2, Alexandra Sneider *1, Weitong Chen 1, Michelle Karl 1, Vishnu Prasath 3, Pei-Hsun Wu 1,2, Gunnar Mattson 3, Denis Wirtz 1,2,4
1Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 2Johns Hopkins Physical Sciences - Oncology Center, Johns Hopkins University, 3Department of Biomedical Engineering, Johns Hopkins University, 4Departments of Oncology and Pathology and Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

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Biology

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation
Yulia Artemenko 1, Peter N. Devreotes 2
1Department of Biological Sciences, SUNY Oswego, 2Department of Cell Biology, Johns Hopkins University School of Medicine

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

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Developmental Biology

Genetic Engineering of Primary Mouse Intestinal Organoids Using Magnetic Nanoparticle Transduction Viral Vectors for Frozen Sectioning
Lingling Xian 1, Lionel Chia 1,5, Dan Georgess 2, Li Luo 1, Shuai Shuai 1, Andrew J. Ewald 3,4, Linda M.S. Resar 1,4,5,6
1Department of Medicine, Division of Hematology, Johns Hopkins University School of Medicine, 2Department of Natural Sciences, School of Arts & Sciences, Lebanese American University, 3Department of Cell Biology, Johns Hopkins University School of Medicine, 4Department of Oncology, Johns Hopkins University School of Medicine, 5Department of Pathology, Johns Hopkins University School of Medicine, 6Institute for Cell Engineering, Johns Hopkins University School of Medicine

We describe step-by-step instructions to: 1) efficiently engineer intestinal organoids using magnetic nanoparticles for lenti- or retroviral transduction, and, 2) generate frozen sections from engineered organoids. This approach provides a powerful tool to efficiently alter gene expression in organoids for investigation of downstream effects.

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Immunology and Infection

Full-Field Optical Coherence Microscopy for Histology-Like Analysis of Stromal Features in Corneal Grafts
Kristina Irsch 1,2,3, Kate Grieve 1,2, Marie Borderie 2, Maëlle Vilbert 1,2,4, Karsten Plamann 4,5, Djida Ghoubay 1,2, Cristina Georgeon 2, Vincent Borderie 1,2
1Vision Institute, Sorbonne University, UM 80 / INSERM, UMR_S 968 / CNRS, UMR_7210, 2Quinze Vingts National Ophthalmology Hospital, 3Laboratory of Ophthalmic Instrument Development, The Wilmer Eye Institute, Johns Hopkins University School of Medicine, 4Laboratory for Optics and Biosciences (LOB), École polytechnique, 5LOA - ENSTA Paris, École polytechnique

We describe use of full-field optical coherence microscopy as a method for high quality assessment of corneal donor stroma. This protocol can be used to identify features indicative of health or disease, and is aimed at improving the screening and selection of donor tissues, and hence the results of keratoplasty.

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Medicine

Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRI—Application in Premanifest Huntington's Disease
Dan Wu 1, Andreia V. Faria 1, Laurent Younes 2,3,4, Christopher A. Ross 5, Susumu Mori 1,6, Michael I. Miller 2,3,7
1The Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, 2Center for Imaging Science, Johns Hopkins University, 3Institute for Computational Medicine, Johns Hopkins University, 4Department of Applied Mathematics and Statistics, Johns Hopkins University, 5Division of Neurobiology, Departments of Psychiatry, Neurology, Neuroscience and Pharmacology, and Program in Cellular and Molecular Medicine, Johns Hopkins University School of Medicine, 6F.M. Kirby Research Center for Functional Brain Imaging, Kennedy Krieger Institute, 7Department of Biomedical Engineering, Johns Hopkins University

This paper describes a statistical model for volumetric MRI data analysis, which identifies the "change-point" when brain atrophy begins in premanifest Huntington's disease. Whole-brain mapping of the change-points is achieved based on brain volumes obtained using an atlas-based segmentation pipeline of T1-weighted images.

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Generation of Three-dimensional Printed Biological Innovative Mold for Optically Transparent Tissue Specimens
Sean J. Miller 1,2,3, Jeffrey D. Rothstein 1,2,3
1Department of Neurology, Johns Hopkins University School of Medicine, 2The Brain Science Institute, Johns Hopkins University School of Medicine, 3Cellular & Molecular Medicine, Johns Hopkins University School of Medicine

Tissue innovative Molds (iMolds) have been developed to reduce specimen movement, structurally support the specimen being imaged, and allow for repeated imaging on precise anatomical locations using optically-transparent samples.

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Immunology and Infection

ExCYT: A Graphical User Interface for Streamlining Analysis of High-Dimensional Cytometry Data
John-William Sidhom 1,2,3, Debebe Theodros 1,2,4, Benjamin Murter 1,2, Jelani C. Zarif 1,2, Sudipto Ganguly 1,2, Drew M. Pardoll 1,2, Alexander Baras 1,2,5
1The Bloomberg~Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University School of Medicine, 2The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, 3Department of Biomedical Engineering, Johns Hopkins University School of Medicine, 4Department of Immunology, Johns Hopkins University School of Medicine, 5Department of Pathology, Johns Hopkins University School of Medicine

ExCYT is a MATLAB-based Graphical User Interface (GUI) that allows users to analyze their flow cytometry data via commonly employed analytical techniques for high-dimensional data including dimensionality reduction via t-SNE, a variety of automated and manual clustering methods, heatmaps, and novel high-dimensional flow plots.

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Immunology and Infection

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo
Line L. Holm 1,2,3, Milica Vukmanovic-Stejic 4, Thomas Blauenfeldt 1, Thomas Benfield 2,3, Peter Andersen 1, Arne N. Akbar 4, Morten Ruhwald 1
1Department of Infectious Disease Immunology, Center for Vaccine Research, Statens Serum Institut, 2Department of Infectious Diseases, Hvidovre Hospital, 3Institute of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, 4Division of Infection and Immunity, University College London

Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

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Biology

Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis
Caleb Sutherland *1, Yu Wang *2, Robert V. Brown *1, Julie Foley 2, Beth Mahler 2, Kyathanahalli S. Janardhan 2,3, Ramesh C. Kovi 2,4, Anton M. Jetten 1
1Immunity, Inflammation, and Disease Laboratory, Division of Intramural Research, National Institute of Environmental Health Sciences, NIH, 2Cellular and Molecular Pathology Branch, Division of the National Toxicology Program, National Institute of Environmental Health Sciences, NIH, 3Integrated Laboratory Systems Inc., 4Experimental Pathology Laboratories Inc.

Here, we describe a protocol for a reproducible laser capture microdissection (LCM) for isolating trabecular meshwork (TM) for downstream RNA analysis. The ability to analyze changes in gene expression in the TM will help in understanding the underlying molecular mechanisms of TM-related ocular diseases.

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Developmental Biology

Semi-automated Production of Hepatocyte Like Cells from Pluripotent Stem Cells
Jose Meseguer-Ripolles 1, Baltasar Lucendo-Villarin 1, Yu Wang 1, David C. Hay 1
1MRC Centre for Regenerative Medicine, University of Edinburgh

This protocol describes a semi-automated approach to produce hepatocyte-like cells from human pluripotent stem cells in a 96 well plate format. This process is rapid and cost-effective, allowing the production of quality assured batches of hepatocyte-like cells for basic and applied human research.

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Neuroscience

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion
Qiao Li 1, Cheng Qian 1, Feng-Quan Zhou 1,2
1Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, 2The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

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Environment

Fabrication of Anisotropic Polymeric Artificial Antigen Presenting Cells for CD8+ T Cell Activation
Elana Ben-Akiva *1, Kelly R. Rhodes *1, Randall A. Meyer 1, Jordan J. Green 2
1Biomedical Engineering, Translational Tissue Engineering Center, Institute for Nanobiotechnology, Johns Hopkins University School of Medicine, 2Biomedical Engineering, Translational Tissue Engineering Center, Institute for Nanobiotechnology, Ophthalmology, Oncology, Neurosurgery, Materials Science and Engineering, Chemical and Biomolecular Engineering, and the Bloomberg-Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University School of Medicine

Here, we present a protocol to quickly and reproducibly generate biologically inspired, biodegradable articifical antigen presenting cells (aAPC) with tunable size, shape, and surface protein presentation for T cell expansion ex vivo or in vivo.

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Developmental Biology

A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome
Ping Xue 1, Zhiqiang Wang 1, Xiaomin Fu 1,2, Junjiang Wang 1,3, Gopika Punchhi 1, Andrew Wolfe 1,4, Sheng Wu 1,4,5
1Department of Pediatrics, Johns Hopkins University School of Medicine, 2Department of Health, Beijing Military General Hospital, 3Southern Medical University, 4Department of Molecular and Cellular Physiology, Johns Hopkins University School of Medicine, 5Department of Gynecology and Obstetrics, Johns Hopkins University School of Medicine

We describe the development of a lean PCOS-like mouse model with dihydrotestosterone pellet to study the pathophysiology of PCOS and the offspring from these PCOS-like dams.

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Medicine

A Metadata Extraction Approach for Clinical Case Reports to Enable Advanced Understanding of Biomedical Concepts
John Harry Caufield 1,2, David A. Liem 1,2,3, Anders O. Garlid 1,2, Yijiang Zhou 4, Karol Watson 1,3, Alex A. T. Bui 1,5,6,7, Wei Wang 1,7,8,9, Peipei Ping 1,2,3,7,8
1The NIH BD2K Center of Excellence in Biomedical Computing, University of California, Los Angeles, 2Department of Physiology, University of California, Los Angeles, 3Department of Medicine/Cardiology, University of California, Los Angeles, 4Department of Cardiology, First Affiliated Hospital, Zhejiang University School of Medicine, 5Department of Radiological Sciences, University of California, Los Angeles, 6Department of Bioengineering, University of California, Los Angeles, 7Scalable Analytics Institute (ScAi), University of California, Los Angeles, 8Department of Bioinformatics, University of California, Los Angeles, 9Department of Computer Science, University of California, Los Angeles

We present a protocol and associated metadata template for the extraction of text describing biomedical concepts in clinical case reports. The structured text values produced through this protocol can support deep analysis of thousands of clinical narratives.

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Biology

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart
Santosh K. Yadav 1, Paras K. Mishra 1,2
1Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, 2Department of Anesthesiology, University of Nebraska Medical Center

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

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Chemistry

Stable DNA Motifs, 1D and 2D Nanostructures Constructed from Small Circular DNA Molecules
Xin Guo 1, Xue-Mei Wang 1, Shou-Jun Xiao 1
1State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University

This article presents a detailed protocol for T4 ligation and denaturing PAGE purification of small circular DNA molecules, annealing and native PAGE analysis of circular tiles, assembling and AFM imaging of 1D and 2D DNA nanostructures, as well as agarose gel electrophoresis and centrifugation purification of finite DNA nanostructures.

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Immunology and Infection

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry
Maria Letizia Giardino Torchia 1, Raffaello Cimbro 2
1Oncology Research, Medimmune, LLC, 2Division of Rheumatology, Johns Hopkins University School of Medicine

Here, we present a flow cytometric protocol to identify CD4+ and CD8+ T cells, γδ T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

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Medicine

Protocol and Guidelines for Point-of-Care Lung Ultrasound in Diagnosing Neonatal Pulmonary Diseases Based on International Expert Consensus
Jing Liu 1,2, Roberto Copetti 3, Erich Sorantin 4, Jovan Lovrenski 5, Javier Rodriguez-Fanjul 6, Dalibor Kurepa 7, Xing Feng 8, Luigi Cattaross 9, Huayan Zhang 10,11, Misun Hwang 12, Tsu F. Yeh 13,14, Yisrael Lipener 7, Abhay Lodha 15, Jia-Qin Wang 16, Hai-Ying Cao 2,17, Cai-Bao Hu 2,18, Guo-Rong Lyu 19, Xin-Ru Qiu 1,2, Li-Qun Jia 20, Xiao-Man Wang 20, Xiao-Ling Ren 1,2, Jiu-Ye Guo 1,2, Yue-Qiao Gao 1,2, Jian-Jun Li 1,2, Ying Liu 1,2, Wei Fu 1,2, Yan Wang 21, Zu-Lin Lu 1,2, Hua-Wei Wang 8, Li-Li Shang 22
1Department of Neonatology and NICU, Beijing Chaoyang District Maternal and Child Healthcare Hospital, 2The Neonatal Lung Ultrasound Training Base, Chinese College of Critical Ultrasound, 3Emergency Department, Cattinara University Hospital, 4Division of Pediatric Radiology, Department of Radiology, Medical University Graz, 5Faculty of Medicine, University of Novi Sad, Radiology Department, Institute for Children and Adolescents Health Care of Vojvodina, 6Pediatric Intensive Care Unit, Pediatric Service Hospital Joan XXIII Tarragona, University Rovira i Virgil, 7Division of Neonatal-Perinatal Medicine, Cohen Children's Medical Center, 8Department of Neonatology, Children's Hospital of Soochow University, 9Department of Neonatology, Udine University Hospital, 10Center for Newborn Care, Guangzhou Women and Children's Medical Center, 11Division of Neonatology, Children's Hospital of Philadelphia, 12Department of Radiology, Children's Hospital of Philadelphia, 13Division of Neonatology and NICU, Cook County Children's Hospital, University of Illinois, 14Division of Neonatology, Department of Pediatrics, Taipei Medical University, 15Department of Pediatrics and Community Health Sciences, University of Calgary, 16Department of Pediatrics, The Third Affiliated Hospital of Xinxiang Medical University, 17Department of Ultrasound, GE Healthcare, 18Intensive Care Unit, Zhejiang Hospital, 19Collaborative Innovation Center for Maternal and Infant Health Service Application Technology, Quanzhou Medical College, 20Department of Ultrasound, Beijing Children's Hospital Affiliated with Capital Medical University, 21Department of Neonatology and NICU, Tai'an City Central Hospital of Shandong Province, 22Department of Intensive Care Unit, The Second Affiliated Hospital of Heilongjiang University of Chinese Medicine

Lung ultrasound is a noninvasive and valuable tool for bedside evaluation of neonatal lung diseases. However, a relative lack of reference standards, protocols and guidelines may limit its application. Here, we aim to develop a standardized neonatal lung ultrasound diagnostic protocol to be used in clinical decision-making.

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Immunology and Infection

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection
Leif S. Anderson 1, Mack B. Reynolds 1, Kathryn R. Rivara 1, Lloyd S. Miller 2, Scott I. Simon 1
1Department of Biomedical Engineering, University of California Davis, 2Department of Dermatology, Johns Hopkins University School of Medicine

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

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Medicine

Cloud-Based Phrase Mining and Analysis of User-Defined Phrase-Category Association in Biomedical Publications
Dibakar Sigdel *1,2, Vincent Kyi *1,2, Aiden Zhang *1, Shaun P. Setty 3, David A. Liem 1,2,4, Yu Shi 5, Xuan Wang 5, Jiaming Shen 5, Wei Wang 1,6,7, JiaWei Han 5, Peipei Ping 1,2,4,6
1The NIH BD2K Center of Excellence in Biomedical Computing, University of California, Los Angeles, 2Department of Physiology, University of California, Los Angeles, 3Department of Pediatric and Adult Congenital Heart Surgery, Miller Children's and Women's Hospital and Long Beach Memorial Hospital, 4Department of Medicine/Cardiology, University of California, Los Angeles, 5NIH BD2K Program Centers of Excellence for Big Data Computing -- KnowEng Center, Department of Computer Science, University of Illinois at Urbana-Champaign (UIUC), 6Scalable Analytics Institute (ScAi), University of California, Los Angeles, 7Department of Computer Science, University of California, Los Angeles

We present a protocol and associated programming code as well as metadata samples to support a cloud-based automated identification of phrases-category association representing unique concepts in user selected knowledge domain in biomedical literature. The phrase-category association quantified by this protocol can facilitate in depth analysis in the selected knowledge domain.

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Immunology and Infection

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium
Julie G. In *1, Jennifer Foulke-Abel *2, Elizabeth Clarke 1, Olga Kovbasnjuk 1
1Department of Internal Medicine, University of New Mexico Health Science Center, 2Department of Medicine, Division of Gastroenterology & Hepatology, Johns Hopkins University School of Medicine

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

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Medicine

Orthotopic Rat Kidney Transplantation: A Novel and Simplified Surgical Approach
Ali R. Ahmadi 1, Le Qi 1, Kenichi Iwasaki 1, Wei Wang 1, Russell N. Wesson 1, Andrew M. Cameron 1, Zhaoli Sun 1
1Department of Surgery, Johns Hopkins University School of Medicine

The purpose of this manuscript and protocol is to explain and demonstrate in detail the surgical procedure of orthotopic kidney transplantation in rats. This method is simplified to achieve the correct perfusion of the donor kidney and shorten the reperfusion time by using the venous and ureteral cuff anastomosis technique.

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Environment

A Uniaxial Compression Experiment with CO2-Bearing Coal Using a Visualized and Constant-Volume Gas-Solid Coupling Test System
Weitao Hou 1, Hanpeng Wang 1, Wei Wang 1, Zhongzhong Liu 1, Qingchuan Li 1
1Research Center of Geotechnical and Structural Engineering, Shandong University

This protocol demonstrates how to prepare a briquette sample and conduct a uniaxial compression experiment with a briquette in different CO2 pressures using a visualized and constant-volume gas-solid coupling test system. It also aims to investigate changes in terms of coal’s physical and mechanical properties induced by CO2 adsorption.

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Biology

Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues
Daniels Konja 1, Cuiting Luo 1, Wai Yan Sun 1, Kangmin Yang 1, Andy W.C. Man 1, Aimin Xu 1, Paul M. Vanhoutte 1, Yu Wang 1
1The State Key Laboratory of Pharmaceutical Biotechnology and the Department of Pharmacology and Pharmacy, University of Hong Kong

The protocol describes the use of wire myography to evaluate the transmural isometric tension of mesenteric arteries isolated from mice, with special consideration of the modulation by factors released from endothelial cells and perivascular adipose tissues.

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Developmental Biology

In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells
Emmanouil Tampakakis 1, Matthew Miyamoto 1, Chulan Kwon 1
1Division of Cardiology, Department Medicine, Johns Hopkins School of Medicine

The purpose of this method is to generate heart field-specific cardiac progenitor cells in vitro in order to study the progenitor cell specification and functional properties, and to generate chamber specific cardiac cells for heart disease modelling.

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Developmental Biology

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells
Balta Lucendo-Villarin 1, Hassan Rashidi 1,2, Sharmin Alhaque 1,3, Lena Fischer 1,4, Jose Meseguer-Ripolles 1, Yu Wang 1, Cliona O'Farrelly 4, Michael Themis 3, David C. Hay 1
1MRC Centre for Regenerative Medicine, University of Edinburgh, 2UCL Great Ormond Street Institute of Child Health, University College London, 3Division of Biosciences, Department of Life Sciences, College of Health and Life Sciences, Brunel University London, 4School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin

This protocol describes an approach to produce hepatospheres from human pluripotent stem cells using a defined culture system and cell self-assembly. This protocol is reproducible in a number of cell lines, cost effective and allows the production of stable human hepatospheres for biomedical application.

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Developmental Biology

Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice
Kyla A. Britson 1,2, Aaron D. Black 2,3, Kathryn R. Wagner 1,2,3,4, Thomas E. Lloyd 1,2,4
1Graduate Program in Cellular and Molecular Medicine, Johns Hopkins University School of Medicine, 2Department of Neurology, Johns Hopkins University School of Medicine, 3Kennedy Krieger Institute, 4Department of Neuroscience, Johns Hopkins University School of Medicine

Complex human diseases can be challenging to model in traditional laboratory model systems. Here, we describe a surgical approach to model human muscle disease through the transplantation of human skeletal muscle biopsies into immunodeficient mice.

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Medicine

Immunoglobulin G N-Glycan Analysis by Ultra-Performance Liquid Chromatography
Di Liu *1, Xizhu Xu *2, Yuejin Li 2, Jie Zhang 1, Xiaoyu Zhang 1, Qihuan Li 1, Haifeng Hou 2, Dong Li 2, Wei Wang 1,2,3, Youxin Wang 1
1Beijing Key Laboratory of Clinical Epidemiology, School of Public Health, Capital Medical University, 2School of Public Health, Shandong First Medical University & Shandong Academy of Medical Sciences, 3School of Medical and Health Sciences, Edith Cowan University

Immunoglobulin G (IgG) N-glycan is characterized using hydrophilic interaction chromatography UPLC. In addition, the structure of IgG N-glycan is clearly separated. Presented here is an introduction to this experimental method so that it can be widely used in research settings.

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Cancer Research

Establishing a Competing Risk Regression Nomogram Model for Survival Data
Lunpo Wu 1,2, Chenyang Ge 3, Hongjuan Zheng 4, Haiping Lin 5, Wei Fu 6, Jianfei Fu 4
1Department of Gastroenterology, Second Affiliated Hospital of Zhejiang University School of Medicine, 2Institute of Gastroenterology, Zhejiang University, 3Department of Colorectal Surgery, Afiliated Jinhua Hospital, Zhejiang University School of Medicine, 4Department of Medical Oncology, Affiliated Jinhua Hospital, Zhejiang University School of Medicine, 5Department of Hepatobiliary Pancreatic Surgery, Affiliated Jinhua Hospital, Zhejiang University School of Medicine, 6Division of Oncology, Johns Hopkins University School of Medicine

Presented here is a protocol to build nomograms based on the Cox proportional hazards regression model and competing risk regression model. The competing method is a more rational method to apply when competing events are present in the survival analysis.

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JoVE Core

International Expert Consensus and Recommendations for Neonatal Pneumothorax Ultrasound Diagnosis and Ultrasound-guided Thoracentesis Procedure
Jing Liu 1,2, Dalibor Kurepa 3, Francesco Feletti 4,5, Almudena Alonso-Ojembarrena 6, Jovan Lovrenski 7, Roberto Copetti 8, Erich Sorantin 9, Javier Rodriguez-Fanjul 10, Karishma Katti 3, Andrea Aliverti 4, Huayan Zhang 11,12, Misun Hwang 13, Tsu F. Yeh 14, Cai-Bao Hu 15, Xing Feng 16, Ru-Xin Qiu 1,2, Jing-Han Chi 17, Li-Li Shang 18, Guo-Rong Lyu 19, Shao-Zheng He 20, Yan-Fen Chai 21, Zhan-Jun Qiu 22, Hai-Ying Cao 2,23, Yue-Qiao Gao 1,2, Xiao-Ling Ren 1,2, Guo Guo 1,24, Li Zhang 1,2, Ying Liu 1,2, Wei Fu 1,2, Zu-Lin Lu 1,2, Hong-Lei Li 1,2
1Department of Neonatology and NICU, Beijing Chaoyang District Maternal and Child Healthcare Hospital, 2The National Neonatal Lung Ultrasound Training Base, 3Division of Neonatal-Perinatal Medicine, Cohen Children's Medical Center, 4Department of Electronics, Information and Bioengineering, Politecnico di Milano, 5Dipartimento di Diagnostica per Immagini, Ausl della Romagna, S. Maria delle Croci Hospital, 6Neonatal Intensive Care Unit, Puerta del Mar University Hospital, 7Faculty of Medicine, University of Novi Sad, Serbia, Institute for Children and Adolescents Health Care of Vojvodina, 8Emergency Department, University Hospital of Cattinara, 9Division of Pediatric Radiology, Department of Radiology, Medical University Graz, 10Pediatric Intensive Care Unit, Pediatric Service Hospital Joan XXIII Tarragona, University Rovira i Virgil, 11Center for Newborn Care, Guangzhou Women and Children's Medical Center, 12Division of Neonatology, Children's Hospital of Philadelphia, 13Section of Neonatal Imaging, Department of Radiology, Children's Hospital of Philadelphia, 14Maternal Child Health Research institute, Taipei Medical University and China Medical University, 15Intensive Care Unit, Zhejiang Hospital, 16Department of Neonatology, Children's Hospital of Soochow University, 17Department of Neonatology and NICU, Bayi Children's Hospital Affiliated to the Seventh Medical Center of Chinese PLA General Hospital, 18Intensive Care Unit, Second Affiliated Hospital of Heilongjiang University of Chinese Medicine, 19Collaborative Innovation Center for Maternal and Infant Health Service Application Technology, Quanzhou Medical College, 20Department of Ultrasound, Second Affiliated Hospital of Fujian Medical University, 21Department of Emergency Medicine, Tianjin Medical University General Hospital, 22Department of Emergency and Critical Care Medicine, Affiliated Hospital of Traditional Chinese Medicine, 23Department of Ultrasound, GE Healthcare, 24The Neonatal Intensive Care Unit, Fifth Medical Center of Chinese PLA General Hospital

Pneumothorax is a common emergency and critical disease in newborn infants that needs rapid, clear diagnosis and timely treatment. Diagnosis and treatment based on chest X-rays are associated with delayed management and radiation damage. Lung ultrasound (US) provides useful guidance for rapid, accurate diagnosis and the precise thoracentesis of pneumothorax.

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Genetics

Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing
Yu Wang 1, Andrew J. H. Smith 1, David C. Hay 1
1MRC Centre for Regenerative Medicine, University of Edinburgh

Here, we describe a detailed method for seamless gene editing in human pluripotent stem cells using a piggyBac-based donor plasmid and the Cas9 nickase mutant. Two point mutations were introduced into exon 8 of the hepatocyte nuclear factor 4 alpha (HNF4α) locus in human embryonic stem cells (hESCs).

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Biology

Hepatic Progenitor Specification from Pluripotent Stem Cells using a Defined Differentiation System
Jose Meseguer-Ripolles 1, Yu Wang 1, Agnes Sorteberg 1, Aishwariya Sharma 2, Nan-Linda Ding 2, Baltasar Lucendo-Villarin 1, Philipp Kramer 2, Charis-Patricia Segeritz 2, David C. Hay 1
1MRC Centre for Regenerative Medicine, University of Edinburgh, 2Research & Development, STEMCELL Technologies Inc

The goal of this article is to provide a standardized approach to induce human hepatic progenitor differentiation from pluripotent stem cells. The development of this procedure with ready-to-use media formulations offer the user a facile system to generate human liver cells for biomedical research and translation.

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Cancer Research

Genome-Wide Analysis of DNA Methylation in Gastrointestinal Cancer
Kiichi Sugimoto 1,2, Hirotaka Momose 2, Tomoaki Ito 1,3, Hajime Orita 3, Koichi Sato 3, Kazuhiro Sakamoto 2, Malcolm V. Brock 1
1Department of Surgery, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, 2Department of Coloproctological Surgery, Juntendo University Faculty of Medicine, 3Department of Surgery, Juntendo University Shizuoka Hospital

Herein, we describe a procedure for genome-wide analysis of DNA methylation in gastrointestinal cancers. The procedure is of relevance to studies that investigate relationships between methylation patterns of genes and factors contributing to carcinogenesis in gastrointestinal cancers.

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Behavior

A Chronic Sleep Fragmentation Model using Vibrating Orbital Rotor to Induce Cognitive Deficit and Anxiety-Like Behavior in Young Wild-Type Mice
Yi Xie 1, Sai-Yue Deng 1, Si-Miao Chen 1, Xue-Jiao Chen 1, Wen-Wen Lai 1, Li-Fang Huang 1, Li Ba 1, Wei Wang 1,2, Feng-Fei Ding 1,3
1Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430000, China, 2Key Laboratory of Neurological Diseases of Chinese Ministry of Education, The School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430000,China, 3Department of Pharmacology, School of Basic Medical Science; State Key Laboratory of Medical Neurobiology, Institutes of Brain Science and Collaborative Innovation Center for Brain Science, Fudan University, Shanghai 200032, China

Presented here is a protocol for chronic sleep fragmentation (CSF) model achieved by an electrically controlled orbital rotor, which could induce confirmed cognitive deficit and anxiety-like behavior in young wild-type mice. This model can be applied to explore the pathogenesis of chronic sleep disturbance and related disorders.

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Medicine

Acupuncture in a Rat Model of Asthma
Dong-Dong Zhou 1, Guang-Quan Zhang 1, Qing-Yi Zhao 1, Mi Cheng 1, Jin Lu 1, Yu Wang 1, Yu-Dong Xu 1, Yan-Jiao Chen 1, Yong-Qing Yang 1, Lei-Miao Yin 1,2
1Laboratory of Molecular Biology, Shanghai Research Institute of Acupuncture and Meridian, Shanghai University of Traditional Chinese Medicine, 2Shanghai Innovation Center of Traditional Chinese Medicine Health Service

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes methods for the fabrication of the high platform, establishes a rat model of asthma and measures changes in respiratory function using a noninvasive and real-time whole-body plethysmography (WBP) system.

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Biology

An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation
Mingxiao Feng 1, Sara Divall 3, Sheng Wu 1,2
1Department of Pediatrics, Johns Hopkins University School of Medicine, 2Department of Physiology, Temple University School of Medicine, 3Department of Pediatrics, Seattle Children’s Hospital

Primary hepatocytes are a valuable tool to study liver response and metabolism in vitro. Utilizing commercially available reagents, an improved time- and labor-efficient protocol for mouse primary hepatocyte isolation was developed.

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Neuroscience

Establishment of a Rat Model of Superior Sagittal-Sinus Occlusion via a Thread-Embolism Method
Weiyan Jiang *1, Congcong Jin *2, Weiming Xu *3, Yingxian Li 4, Yinghong Lin 5, Shengxiang Liang 6,7, Wei Wang 8
1Department of Clinical Laboratory, the Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University, 2Rehabilitation Department, Wenzhou Hospital of Integrated Traditional Chinese and Western Medicine, 3Department of Neurosurgery, the Hospital of Changle, 4Pediatrics Ward 2, Shouguang Hospital of Traditional Chinese Medicine, 5Fujian University of Traditional Chinese Medicine, 6National-Local Joint Engineering Research Center of Rehabilitation Medicine Technology, Fujian University of Traditional Chinese Medicine, 7Rehabilitation Industry Institute, Fujian University of Traditional Chinese Medicine, 8Department of Neurosurgery, the First Affiliated Hospital of Wenzhou Medical University

Here, we establish a novel Sprague-Dawley (SD) rat model of superior sagittal sinus (SSS) thrombosis via a thread-embolization method, and the stability and reliability of the model were verified.

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Cancer Research

A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate
Shun Ishiyama 1,2,3,4, Casey Kissel 5, Xin Guo 1, Alexis Howard 6, Harumi Saeki 7, Tomoaki Ito 8, Polina Sysa-Shah 9, Hajime Orita 10, Kazuhiro Sakamoto 4, Kathleen Gabrielson 1,2
1Department of Molecular and Comparative Pathobiology, The Johns Hopkins University School of Medicine, 2Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, 3Department of Surgery, Johns Hopkins University School of Medicine, 4Department of Coloproctological Surgery, Juntendo University Faculty of Medicine, 5Program for Comparative Medicine, Gene Therapy Program, Perelman School of Medicine, University of Pennsylvania, 6Tuskegee College of Veterinary Medicine, 7Department of Human Pathology, Juntendo University Faculty of Medicine, 8Department of Surgery, Juntendo University Shizuoka Hospital, Juntendo University School of Medicine, 9Department of Urology, The Johns Hopkins School of Medicine, 10Department of Gastroenterology and Minimally Invasive Surgery, Juntendo University Faculty of Medicine

Here, a syngeneic orthotopic implantation followed by an amputation procedure of the osteosarcoma with spontaneous pulmonary metastasis that can be used for preclinical investigation of metastasis biology and development of novel therapeutics is described.

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Neuroscience

Multiplexed Analysis of Retinal Gene Expression and Chromatin Accessibility Using scRNA-Seq and scATAC-Seq
Kurt Weir *1, Patrick Leavey *1, Clayton Santiago 1, Seth Blackshaw 1,2,3,4,5,6
1Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, 2Department of Psychiatry and Behavioral Science, The Johns Hopkins Hospital, 3Department of Ophthalmology, The Johns Hopkins Hospital, 4Department of Neurology, The Johns Hopkins Hospital, 5Institute for Cell Engineering, Johns Hopkins University School of Medicine, 6Kavli Neuroscience Discovery Institute, Johns Hopkins University School of Medicine

Here, the authors showcase the utility of MULTI-seq for phenotyping and subsequent paired scRNA-seq and scATAC-seq in characterizing the transcriptomic and chromatin accessibility profiles in retina.

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Biochemistry

Ganglioside Extraction, Purification and Profiling
Mitchell J. Porter *1, Gao-Lan Zhang *1, Ronald L. Schnaar 1,2
1Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, 2Department of Neuroscience, Johns Hopkins University School of Medicine

Gangliosides are sialic acid-bearing glycosphingolipids that are particularly abundant in the brain. Their amphipathic nature requires organic/aqueous extraction and purification techniques to ensure optimal recovery and accurate analyses. This article provides overviews of analytic and preparative scale ganglioside extraction, purification, and thin layer chromatography analysis.

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Neuroscience

Nuclei Isolation and Super-Resolution Structured Illumination Microscopy for Examining Nucleoporin Alterations in Human Neurodegeneration
Alyssa N. Coyne 1,2, Jeffrey D. Rothstein 1,2
1Brain Science Institute, Johns Hopkins University School of Medicine, 2Department of Neurology, Johns Hopkins University School of Medicine

This protocol describes an optimized workflow for nuclei isolation and super-resolution structured illumination microscopy to evaluate individual nucleoporins within the nucleoplasm and NPCs in induced pluripotent stem cell derived neurons and postmortem human tissues.

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JoVE Journal

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis
Chi-Ju Kim *1,2, Morgan D. Kuczler *1, Liang Dong 1,3, Junyoung Kim 2,4, Sarah R. Amend 1, Yoon-Kyoung Cho 2,4, Kenneth J. Pienta 1
1The Brady Urological Institute, Johns Hopkins University School of Medicine, 2Department of Biomedical Engineering, School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), 3Department of Urology, Renji Hospital, Shanghai Jiao Tong University School of Medicine, 4Center for Soft and Living Matter, Institute for Basic Science (IBS)

Extracellular vesicles (EVs) contribute to cellular biology and intercellular communications. There is a need for practical assays to visualize and quantify EVs uptake by the cells. The current protocol proposes the EV uptake assay by utilizing three-dimensional fluorescence imaging via confocal microscopy, following EV isolation by a nano-filtration-based microfluidic device.

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Biology

Development of a Cell Co-Culture Model to Mimic Cardiac Ischemia/Reperfusion In Vitro
Zhu Li 1, Matthew J. W. Hampton 1, Matthew B. Barajas 1, Matthias L. Riess 1,2,3
1Department of Anesthesiology, Vanderbilt University Medical Center, 2Anesthesiology, TVHS VA Medical Center, 3Department of Pharmacology, Vanderbilt University

Spatial distance is a key parameter in assessing hypoxia/reoxygenation injury in a co-culture model of separate endothelial and cardiomyocyte cell layers, suggesting, for the first time, that optimizing the co-culture spatial environment is necessary to provide a favorable in vitro model for testing the role of endothelial cells in cardiomyocyte protection.

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Immunology and Infection

Plaquing of Herpes Simplex Viruses
Lauren A. Sadowski *1, Gregory M. Lesko *1, Chad Suissa *1, Rista Upadhyay *1,2, Prashant J. Desai 3, Barry J. Margulies 1,4
1Towson University Herpes Virus Lab, Department of Biological Sciences, Towson University, 2Towson University Department of Chemistry, Towson University, 3Department of Oncology, Johns Hopkins University School of Medicine, 4Molecular Biology, Biochemistry, and Bioinformatics Program, Towson University

Plaquing is a routine method used to quantify live viruses in a population. Though plaquing is frequently taught in various microbiology curricula with bacteria and bacteriophages, plaquing of mammalian viruses is more complex and time-consuming. This protocol describes the procedures that function reliably for regular work with herpes simplex viruses.

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Cancer Research

Digital Spatial Profiling for Characterization of the Microenvironment in Adult-Type Diffusely Infiltrating Glioma
Nishika Karbhari 1, Rachael Barney 2, Scott Palisoul 2, Jennifer Hong 3, Chun-Chieh Lin 2, George Zanazzi 2,4
1Department of Neurology, Dartmouth-Hitchcock Medical Center, 2Department of Pathology and Laboratory Medicine, Dartmouth-Hitchcock Medical Center, 3Department of Neurosurgery, Dartmouth-Hitchcock Medical Center, 4Dartmouth Cancer Center, Dartmouth-Hitchcock Medical Center

Proteomic dysregulation plays an important role in the spread of diffusely infiltrating gliomas, but several relevant proteins remain unidentified. Digital spatial processing (DSP) offers an efficient, high-throughput approach for characterizing the differential expression of candidate proteins that may contribute to the invasion and migration of infiltrative gliomas.

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Biology

Evaluation of Fatty Acid Oxidation in Cultured Bone Cells
Soohyun P. Kim 1, Zhu Li 1, Ryan C. Riddle 1,2
1Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, 2Research and Development Service, Baltimore Veterans Administration Medical Center

The present protocol describes an assay to assess the capacity for fatty acid oxidation in cultures of primary bone cells or relevant cell lines.

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JoVE Core

Experimental Model to Evaluate Resolution of Pneumonia
Andres Villabona-Rueda 1, Daniel Wang 2, Franco R. D’Alessio 1
1Division of Pulmonary Critical Care Medicine, Johns Hopkins University School of Medicine, 2Engineering Management, Whiting School of Engineering, Johns Hopkins University

This manuscript describes the establishment of an infectious model of pneumonia in mice and the respective characterization of injury resolution along with methods for growing bacteria and intratracheal instillation. A novel approach using high-dimensional flow cytometry to evaluate the immune landscape is also described.

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Immunology and Infection

High-Speed Human Temporal Bone Sectioning for the Assessment of COVID-19-Associated Middle Ear Pathology
Nicholas S. Andresen 1, Megan K. Wood 2, Daniela Čiháková 3, C. Matthew Stewart 1
1Department of Otolaryngology - Head & Neck Surgery, Johns Hopkins University School of Medicine, 2W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, 3Department of Pathology, Johns Hopkins University School of Medicine

This article describes a technique for rapid human temporal bone sectioning that utilizes a microsaw with twin diamond blades to generate thin slices for rapid decalcification and analysis of temporal bone immunohistochemistry.

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Cancer Research

Implementation of Minimally Invasive Brain Tumor Resection in Rodents for High Viability Tissue Collection
Safwan Alomari 1, Jayanidhi Kedda 1, Adarsha P. Malla 2,3, Victor Pacis 1, Pavlos Anastasiadis 2,3, Su Xu 4, Emylee McFarland 2,3, Lilia Sukhon 1, Bruno Gallo 5, Jordina Rincon-Torroella 1, Netanel Ben-Shalom 1, Heather M. Ames 3,6, Henry Brem 1,7, Graeme F. Woodworth 2,3, Betty Tyler 1
1Department of Neurosurgery, Johns Hopkins University School of Medicine, 2Department of Neurosurgery, University of Maryland School of Medicine, 3Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland School of Medicine, 4Diagnostic Radiology and Nuclear Medicine, University of Maryland School of Medicine, 5Pontifical Catholic University of Parana, 6Department of Pathology, University of Maryland School of Medicine, 7Departments of Ophthalmology, Oncology and Biomedical Engineering, Johns Hopkins University School of Medicine

The present protocol describes a standardized resection of brain tumors in rodents through a minimally invasive approach with an integrated tissue preservation system. This technique has implications for accurately mirroring the standard of care in rodent and other animal models.

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Biology

Field-Deployable Candidatus Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a
Min Li *1, Hongkun Qin *1, Yunfei Long 1, Meiqin Cheng 1, Ling Li 1, Aijun Huang 2, Nian Wang 3, Shuo Duan 1
1China-USA Citrus Huanglongbing Joint Laboratory, National Navel Orange Engineering Research Center, Gannan Normal University, 2Department of Biological Sciences, Gannan Normal University, 3Citrus Research and Education Center, Department of Microbiology and Cell Science, University of Florida - Institute of Food and Agricultural Sciences

In this work, a rapid, sensitive, and portable detection method for Candidatus Liberibacter asiaticus based on recombinase polymerase amplification combined with CRISPR-Cas12a was developed.

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Immunology and Infection

A Mouse Model of Orotracheal Intubation and Ventilated Lung Ischemia Reperfusion Surgery
Wen-I Liao *1, Daisuke Maruyama *1, Farzaneh Kianian 1,2, Christine Tat 1, Xiaoli Tian 1, Judith Hellman 1, Jeffrey M Dodd-o 3, Arun Prakash 1
1Department of Anesthesia and Perioperative Care, University of California San Francisco and San Francisco General Hospital, 2Department of Physiology, School of Medicine, Tehran University of Medical Sciences, 3Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine

A mouse surgical model to create left lung ischemia reperfusion (IR) injury while maintaining ventilation and avoiding hypoxia.

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Cancer Research

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice
Rachel Vistein 1, Briana Winer 2, Stephanie Myers 1,3, Juliane Liberto 4, Shun Ishiyama 1,5,6, Xin Guo 1, Harumi Saeki 7, Tian-Li Wang 4,8, Ie-Ming Shih 2,4,8, Kathleen Gabrielson 1,4,8
1Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, 2Department of Obstetrics and Gynecology, Johns Hopkins University School of Medicine, 3Texas Tech University School of Veterinary Medicine, 4Department of Pathology, Johns Hopkins University School of Medicine, 5Department of Surgery, Johns Hopkins University School of Medicine, 6Department of Coloproctological Surgery, Juntendo University School of Medicine, 7Department of Human Pathology, Juntendo University School of Medicine, 8Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University

This protocol describes a method for monitoring the progression of morphological changes over time in the uterus in an inducible mouse model of endometrial cancer using ultrasound imaging with correlation to gross and histological changes.

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Biochemistry

Detection and Quantification of Calcitonin Gene-Related Peptide (CGRP) in Human Plasma Using a Modified Enzyme-Linked Immunosorbent Assay
Pavan S. Krishnan 1,2, Fernando T. Zamuner 1,3, Carolyn M. Jenks 1, Johnny Y. Xie 4, Lisa Zhang 5, Mohammed Lehar 1, Neal S. Fedarko 6, Mariana Brait 1,3, John P. Carey 1
1Department of Otolaryngology-Head & Neck Surgery, Johns Hopkins University School of Medicine, 2Virginia Commonwealth University School of Medicine, 3Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, 4Department of Otolaryngology-Head and Neck Surgery, University of Michigan Medical School, 5Department of Otolaryngology-Head and Neck Surgery, The Ohio State University College of Medicine, 6ICTR Clinical Research Core Laboratory, Johns Hopkins University School of Medicine

Published data pertaining to calcitonin gene-related peptide (CGRP) concentrations in human plasma are inconsistent. These inconsistencies may be due to the lack of a standardized, validated methodology to quantify this neuropeptide. Here, we describe a validated enzyme-linked immunosorbent assay (ELISA) protocol to purify and quantify CGRP in human plasma.

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Bioengineering

Disruption of the Blood-Spinal Cord Barrier Using Low-Intensity Focused Ultrasound in a Rat Model
Meghana Bhimreddy 1, Denis Routkevitch 1,2,3, Andrew M. Hersh 1, Ali Mohammadabadi 1,3, Arjun K. Menta 1, Kelly Jiang 1, Carly Weber-Levine 1, A. Daniel Davidar 1, Joshua Punnoose 1,2,3, Kelley M. Kempski Leadingham 1,3, Joshua C. Doloff 2, Betty Tyler 1, Nicholas Theodore 1,3, Amir Manbachi 1,2,3,4,5,6
1Department of Neurosurgery, Johns Hopkins University School of Medicine, 2Department of Biomedical Engineering, Johns Hopkins University, 3HEPIUS Innovation Laboratory, Johns Hopkins University School of Medicine, 4Department of Electrical Engineering and Computer Science, Johns Hopkins University, 5Department of Mechanical Engineering, Johns Hopkins University, 6Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University

Disruption of the blood-spinal cord barrier (BSCB) can be successfully achieved with the intravenous administration of microbubbles and the application of low-intensity focused ultrasound (LIFU). This protocol details the opening of the BSCB using LIFU in a rodent model, including equipment setup, microbubble injection, target localization, and BSCB disruption visualization.

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Bioengineering

Sonodynamic Therapy for the Treatment of Glioblastoma Multiforme in a Mouse Model Using a Portable Benchtop Focused Ultrasound System
Griffin Mess 1,2, Taylor Anderson 1,2, Shivani Kapoor 3, Rasika Thombre 1,2, Ruixing Liang 4, Emre Derin 1, Kelley M. Kempski-Leadingham 1, Santosh K. Yadav 5, Betty Tyler 1, Amir Manbachi 1,2,4,6,7
1Department of Neurosurgery, Johns Hopkins University School of Medicine, 2Department of Biomedical Engineering, Johns Hopkins University, 3Krieger School of Arts and Sciences, Johns Hopkins University, 4Department of Electrical Engineering and Computer Science, Johns Hopkins University, 5Department of Radiology, Cancer Imaging Division, Johns Hopkins University School of Medicine, 6Department of Mechanical Engineering, Johns Hopkins University, 7Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine

Here, we describe a protocol that details how to perform sonodynamic therapy in an in vivo mouse glioblastoma model using magnetic resonance-guided focused ultrasound.

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Focused Ultrasound Neuromodulation of Human <em>In Vitro</em> Neural Cultures in Multi-Well Microelectrode Arrays

Focused Ultrasound Neuromodulation of Human In Vitro Neural Cultures in Multi-Well Microelectrode Arrays
Ruixing Liang 1,2, Griffin Mess 2,3, Joshua Punnoose 2,3, Kelley M. Kempski Leadingham 2,3, Constantin Smit 2,3, Nitish Thakor 1,4, Christa W. Habela 3, Betty Tyler 3, Yousef Salimpour 3, Amir Manbachi 1,2,3,4,5,6
1Electrical and Computer Engineering, Johns Hopkins University, 2HEPIUS Innovation Laboratory, Johns Hopkins University School of Medicine, 3Neurosurgery, Johns Hopkins University School of Medicine, 4Biomedical Engineering, Johns Hopkins University School of Medicine, 5Mechanical Engineering, Johns Hopkins University, 6Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine

Here, we present a protocol for using a high-throughput system that enables the monitoring and quantification of the neuromodulatory effects of focused ultrasound on human-induced pluripotent stem cell (HiPSC) neurons.

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Medicine

Hickman Catheter Use for Long-Term Vascular Access in a Preclinical Swine Model
Alisa O. Girard 1, Tessa E. Muss 1, Amanda H. Loftin 1, Richa Kalsi 1, Amy K. Bodine 1, Christopher D. Lopez 1, Georg J. Furtmüller 1, Joanna W. Etra 1, Jessica Izzi 2, Jessica Plunkard 2, Mallory G. Brown 2, Byoung Chol Oh 1, Gerald Brandacher 1
1Department of Plastic and Reconstructive Surgery, Vascularized Composite Allotransplantation (VCA) Laboratory, Johns Hopkins University School of Medicine, 2Department of Molecular and Comparative Pathobiology, Research Animal Resources, Johns Hopkins University School of Medicine

A reliable and reproducible approach for the insertion and maintenance of a tunneled Hickman catheter for long-term vascular access in swine is described. Placement of a central venous catheter allows for convenient daily sampling of whole blood from awake animals and intravenous administration of medication and fluids.

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Transpupillary-Guided Trans-Scleral Transplantation of Subretinal Grafts in a Retinal Degeneration Mouse Model

Transpupillary-Guided Trans-Scleral Transplantation of Subretinal Grafts in a Retinal Degeneration Mouse Model
Ying V. Liu 1, Kang V. Li 1, Zhuolin Li 1, Yuchen Lu 1, Minda M. McNally 1, Edward P. Esposito 1, Kanza Aziz 1, Mandeep S. Singh 1,2
1Wilmer Eye Institute, Johns Hopkins University School of Medicine, 2Department of Genetic Medicine, Johns Hopkins University School of Medicine

This protocol presents a transpupillary vision-guided trans-scleral approach to safely and precisely deliver subretinal cellular grafts, with a low rate of surgical complications, in mouse recipients with or without retinal degeneration.

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Biology

Computational Analysis Tutorial for Chimeric Small Noncoding RNA: Target RNA Sequencing Libraries
Sreenivas Eadara 1, Xinbei Li 1, Emily A. Eiss 1, Mollie K. Meffert 1,2
1Department of Biological Chemistry, Johns Hopkins University School of Medicine, 2Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine

Here, we present a protocol demonstrating the installation and use of a bioinformatics pipeline to analyze chimeric RNA sequencing data used in the study of in vivo RNA:RNA interactions.

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Neuroscience

Hybridization Chain Reaction RNA Whole-Mount Fluorescence In situ Hybridization of Chemosensory Genes in Mosquito Olfactory Appendages
Joshua I. Raji 1, Christopher J. Potter 1
1The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine

The article describes the methods and reagents necessary to perform hybridization chain reaction RNA whole-mount fluorescence in situ hybridization (HCR RNA WM-FISH) to reveal insights into the spatial and cellular resolution of chemosensory receptor genes in the mosquito antenna and maxillary palp.

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Cancer Research

Use of Micropipette-Guided Drug Administration as an Alternative Method to Oral Gavage in Rodent Models
Angélica Cruz Lebrón 1, Emily Blackwell 1,2, Pedro Balbuena Almodóvar 1, Tasnim Syakirah Faiez 1, Luke A. Mummert 1, Karen S. Sfanos 1,3,4
1Department of Pathology, Johns Hopkins University School of Medicine, 2Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, 3Sidney Kimmel Comprehensive Cancer Center, 4Department of Urology, Johns Hopkins University School of Medicine

Here, we describe micropipette-guided drug administration (MDA) as an alternative method to oral gavage that incentivizes the research animal to ingest treatments readily with minimal stress and discomfort.

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Determining the Serum Stability of Human Adenosine Deaminase 1 Enzyme

Determining the Serum Stability of Human Adenosine Deaminase 1 Enzyme
Yusef Haikal 1, Maria Rain Jennings 2, Johnathan Nguyen 1, John J. Blazeck 1
1School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, 2Johns Hopkins University School of Medicine

In this article, we detail methods to characterize an enzyme's ability to retain function when incubated at 37 °C in human serum, a pharmacological property referred to as its serum stability. This ability may be a key factor in predicting an enzyme's pharmacokinetic profile and its suitability for therapeutic use.

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