Johns Hopkins University School of Medicine 79 articles published in JoVE Neuroscience Focused Ultrasound Neuromodulation of Human In Vitro Neural Cultures in Multi-Well Microelectrode Arrays Ruixing Liang1,2, Griffin Mess2,3, Joshua Punnoose2,3, Kelley M. Kempski Leadingham2,3, Constantin Smit2,3, Nitish Thakor1,4, Christa W. Habela3, Betty Tyler3, Yousef Salimpour3, Amir Manbachi1,2,3,4,5,6 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. Neuroscience Transpupillary-Guided Trans-Scleral Transplantation of Subretinal Grafts in a Retinal Degeneration Mouse Model Ying V. Liu1, Kang V. Li1, Zhuolin Li1, Yuchen Lu1, Minda M. McNally1, Edward P. Esposito1, Kanza Aziz1, Mandeep S. Singh1,2 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. Biology Computational Analysis Tutorial for Chimeric Small Noncoding RNA: Target RNA Sequencing Libraries Sreenivas Eadara1, Xinbei Li1, Emily A. Eiss1, Mollie K. Meffert1,2 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. Neuroscience Hybridization Chain Reaction RNA Whole-Mount Fluorescence In situ Hybridization of Chemosensory Genes in Mosquito Olfactory Appendages Joshua I. Raji1, Christopher J. Potter1 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. Biochemistry Detection and Quantification of Calcitonin Gene-Related Peptide (CGRP) in Human Plasma Using a Modified Enzyme-Linked Immunosorbent Assay Pavan S. Krishnan1,2, Fernando T. Zamuner1,3, Carolyn M. Jenks1, Johnny Y. Xie4, Lisa Zhang5, Mohammed Lehar1, Neal S. Fedarko6, Mariana Brait1,3, John P. Carey1 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. Medicine Hickman Catheter Use for Long-Term Vascular Access in a Preclinical Swine Model Alisa O. Girard1, Tessa E. Muss1, Amanda H. Loftin1, Richa Kalsi1, Amy K. Bodine1, Christopher D. Lopez1, Georg J. Furtmüller1, Joanna W. Etra1, Jessica Izzi2, Jessica Plunkard2, Mallory G. Brown2, Byoung Chol Oh1, Gerald Brandacher1 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. Bioengineering Disruption of the Blood-Spinal Cord Barrier Using Low-Intensity Focused Ultrasound in a Rat Model Meghana Bhimreddy1, Denis Routkevitch1,2,3, Andrew M. Hersh1, Ali Mohammadabadi1,3, Arjun K. Menta1, Kelly Jiang1, Carly Weber-Levine1, A. Daniel Davidar1, Joshua Punnoose1,2,3, Kelley M. Kempski Leadingham1,3, Joshua C. Doloff2, Betty Tyler1, Nicholas Theodore1,3, Amir Manbachi1,2,3,4,5,6 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. Cancer Research Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice Rachel Vistein1, Briana Winer2, Stephanie Myers1,3, Juliane Liberto4, Shun Ishiyama1,5,6, Xin Guo1, Harumi Saeki7, Tian-Li Wang4,8, Ie-Ming Shih2,4,8, Kathleen Gabrielson1,4,8 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. Bioengineering Sonodynamic Therapy for the Treatment of Glioblastoma Multiforme in a Mouse Model Using a Portable Benchtop Focused Ultrasound System Griffin Mess1,2, Taylor Anderson1,2, Shivani Kapoor3, Rasika Thombre1,2, Ruixing Liang4, Emre Derin1, Kelley M. Kempski-Leadingham1, Santosh K. Yadav5, Betty Tyler1, Amir Manbachi1,2,4,6,7 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. Immunology and Infection Full-Field Optical Coherence Microscopy for Histology-Like Analysis of Stromal Features in Corneal Grafts Kristina Irsch1,2,3, Kate Grieve1,2, Marie Borderie2, Maëlle Vilbert1,2,4, Karsten Plamann4,5, Djida Ghoubay1,2, Cristina Georgeon2, Vincent Borderie1,2 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. Immunology and Infection A Mouse Model of Orotracheal Intubation and Ventilated Lung Ischemia Reperfusion Surgery Wen-I Liao*1, Daisuke Maruyama*1, Farzaneh Kianian1,2, Christine Tat1, Xiaoli Tian1, Judith Hellman1, Jeffrey M Dodd-o3, Arun Prakash1 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. Immunology and Infection Flow Cytometry-Based Quantification and Analysis of Myocardial B-Cells Kevin C. Bermea1, Sylvie T. Rousseau1, Luigi Adamo1 1Division of Cardiology, Department of Medicine, The Johns Hopkins University School of Medicine Here we report a protocol for the quantification and differentiation of myocardial B-lymphocytes based on their location in the intravascular or endothelial space using flow cytometry. Immunology and Infection High-Speed Human Temporal Bone Sectioning for the Assessment of COVID-19-Associated Middle Ear Pathology Nicholas S. Andresen1, Megan K. Wood2, Daniela Čiháková3, C. Matthew Stewart1 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. Cancer Research Implementation of Minimally Invasive Brain Tumor Resection in Rodents for High Viability Tissue Collection Safwan Alomari1, Jayanidhi Kedda1, Adarsha P. Malla2,3, Victor Pacis1, Pavlos Anastasiadis2,3, Su Xu4, Emylee McFarland2,3, Lilia Sukhon1, Bruno Gallo5, Jordina Rincon-Torroella1, Netanel Ben-Shalom1, Heather M. Ames3,6, Henry Brem1,7, Graeme F. Woodworth2,3, Betty Tyler1 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. Biology Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes Mabel Taracena1,2, Catherine Hunt1, Pamela Pennington3, Deborah Andrew4,5, Marcelo Jacobs-Lorena5,6, Ellen Dotson1, Michael Wells5,7,8 1Division of Parasitic Diseases and Malaria, Entomology Branch, Centers for Disease Control and Prevention, 2Department of Entomology, Cornell University, 3Centro de Estudios en Biotecnologia, Universidad del Valle de Guatemala, 4Department of Cell Biology, Johns Hopkins School of Medicine, 5Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, 6Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health and Malaria Research Institute, 7Department of Cell Biology, Johns Hopkins School of Medicine, 8Biomedical Sciences Department, Idaho College of Osteopathic Medicine The oral administration of dsRNA produced by bacteria, a delivery method for RNA interference (RNAi) that is routinely used in Caenorhabditis elegans, was successfully applied here to adult mosquitoes. Our method allows for robust reverse genetics studies and transmission-blocking vector studies without the use of injection. Biology Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis Chi-Ju Kim*1,2, Morgan D. Kuczler*1, Liang Dong1,3, Junyoung Kim2,4, Sarah R. Amend1, Yoon-Kyoung Cho2,4, Kenneth J. Pienta1 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. 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. Desai3, Barry J. Margulies1,4 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. Biology An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation Mingxiao Feng1, Sara Divall3, Sheng Wu1,2 1Department of Pediatrics, Johns Hopkins University School of Medicine, 2Department of Physiology, Temple University School of Medicine, 3 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. Biology Dissection and Immunostaining of Larval Salivary Glands from Anopheles gambiae Mosquitoes Michelle Z. Chiu1,2, Steven Lannon1, Marisol Luchetti1, Michael B. Wells3, Deborah J. Andrew1 1Department of Cell Biology, The Johns Hopkins University School of Medicine, 2Tufts School of Medicine, 3Department of Biomedical Sciences, Idaho College of Osteopathic Medicine The adult mosquito salivary gland (SG) is required for the transmission of all mosquito-borne pathogens to their human hosts, including viruses and parasites. This video demonstrates efficient isolation of the SGs from the larval (L4) stage Anopheles gambiae mosquitoes and preparation of the L4 SGs for further analysis. Cancer Research A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate Shun Ishiyama1,2,3,4, Casey Kissel5, Xin Guo1, Alexis Howard6, Harumi Saeki7, Tomoaki Ito8, Polina Sysa-Shah9, Hajime Orita10, Kazuhiro Sakamoto4, Kathleen Gabrielson1,2 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. Biochemistry Ganglioside Extraction, Purification and Profiling Mitchell J. Porter*1, Gao-Lan Zhang*1, Ronald L. Schnaar1,2 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. Neuroscience Multiplexed Analysis of Retinal Gene Expression and Chromatin Accessibility Using scRNA-Seq and scATAC-Seq Kurt Weir*1, Patrick Leavey*1, Clayton Santiago1, Seth Blackshaw1,2,3,4,5,6 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. Cancer Research Establishing a Competing Risk Regression Nomogram Model for Survival Data Lunpo Wu1,2, Chenyang Ge3, Hongjuan Zheng4, Haiping Lin5, Wei Fu6, Jianfei Fu4 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. Cancer Research Genome-Wide Analysis of DNA Methylation in Gastrointestinal Cancer Kiichi Sugimoto1,2, Hirotaka Momose2, Tomoaki Ito1,3, Hajime Orita3, Koichi Sato3, Kazuhiro Sakamoto2, Malcolm V. Brock1 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. Bioengineering Design of a Biocompatible Drug-Eluting Tracheal Stent in Mice with Laryngotracheal Stenosis Madhavi Duvvuri1, Kevin Motz2, Hsiu-Wen Tsai2, Ioan Lina2, Dacheng Ding2, Andrew Lee2, Alexander T. Hillel2 1Department of General Surgery, University of California, San Francisco, 2Department of Otolaryngology Head and Neck Surgery, Johns Hopkins School of Medicine Laryngotracheal stenosis results from pathologic scar deposition that critically narrows the tracheal airway and lacks effective medical therapies. Using a PLLA-PCL (70% poly-L-lactide and 30% polycaprolactone) stent as a local drug delivery system, potential therapies aimed at decreasing scar proliferation in the trachea can be studied. Bioengineering Generation of Heterogeneous Drug Gradients Across Cancer Populations on a Microfluidic Evolution Accelerator for Real-Time Observation Ke-Chih Lin1, Gonzalo Torga2, Yusha Sun1, Kenneth J. Pienta2, James C. Sturm1, Robert H. Austin1 1Princeton University, 2Johns Hopkins Medical Institute We present a microfluidic cancer-on-chip model, the "Evolution Accelerator" technology, which provides a controllable platform for long-term real-time quantitative studies of cancer dynamics within well-defined environmental conditions at the single-cell level. This technology is expected to work as an in vitro model for fundamental research or pre-clinical drug development. Developmental Biology Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice Kyla A. Britson1,2, Aaron D. Black2,3, Kathryn R. Wagner1,2,3,4, Thomas E. Lloyd1,2,4 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. Developmental Biology In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells Emmanouil Tampakakis1, Matthew Miyamoto1, Chulan Kwon1 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. Developmental Biology Genetic Engineering of Primary Mouse Intestinal Organoids Using Magnetic Nanoparticle Transduction Viral Vectors for Frozen Sectioning Lingling Xian1, Lionel Chia1,5, Dan Georgess2, Li Luo1, Shuai Shuai1, Andrew J. Ewald3,4, Linda M.S. Resar1,4,5,6 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. Medicine Orthotopic Rat Kidney Transplantation: A Novel and Simplified Surgical Approach Ali R. Ahmadi1, Le Qi1, Kenichi Iwasaki1, Wei Wang1, Russell N. Wesson1, Andrew M. Cameron1, Zhaoli Sun1 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. 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 Clarke1, Olga Kovbasnjuk1 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. Neuroscience Localization of the Locus Coeruleus in the Mouse Brain Katharina Schmidt1, Bilal Bari2, Martina Ralle3, Clorissa Washington-Hughes1, Abigael Muchenditsi1, Evan Maxey4, Svetlana Lutsenko1 1Department of Physiology, Johns Hopkins University, School of Medicine, 2Department of Neuroscience, Johns Hopkins University, 3Department of Molecular and Medical Genetics, OHSU, 4X-ray science division, Advanced Photon Source, Argonne National Laboratory The locus coeruleus is a small cluster of neurons involved in a variety of physiological processes. Here, we describe a protocol to prepare mouse brain sections for studies of proteins and metals in this nucleus. Immunology and Infection Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry Maria Letizia Giardino Torchia1, Raffaello Cimbro2 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. Immunology and Infection A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection Leif S. Anderson1, Mack B. Reynolds1, Kathryn R. Rivara1, Lloyd S. Miller2, Scott I. Simon1 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. Immunology and Infection ExCYT: A Graphical User Interface for Streamlining Analysis of High-Dimensional Cytometry Data John-William Sidhom1,2,3, Debebe Theodros1,2,4, Benjamin Murter1,2, Jelani C. Zarif1,2, Sudipto Ganguly1,2, Drew M. Pardoll1,2, Alexander Baras1,2,5 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. Biochemistry A Rat Methyl-Seq Platform to Identify Epigenetic Changes Associated with Stress Exposure Jenny L. Carey1, Olivia H. Cox1, Fayaz Seifuddin1, Leonard Marque1, Kellie L.K. Tamashiro1, Peter P. Zandi1,3, Gary S. Wand1,2, Richard S. Lee1 1Departments of Psychiatry and Behavioral Sciences, Johns Hopkins School of Medicine, 2Department of Medicine, Johns Hopkins School of Medicine, 3Department of Mental Health, Johns Hopkins School of Public Health Here, we describe the protocol and implementation of Methyl-Seq, an epigenomic platform, using a rat model to identify epigenetic changes associated with chronic stress exposure. Results demonstrate that the rat Methyl-Seq platform is capable of detecting methylation differences that arise from stress exposure in rats. Environment Fabrication of Anisotropic Polymeric Artificial Antigen Presenting Cells for CD8+ T Cell Activation Elana Ben-Akiva*1, Kelly R. Rhodes*1, Randall A. Meyer1, Jordan J. Green2 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. Developmental Biology A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome Ping Xue1, Zhiqiang Wang1, Xiaomin Fu1,2, Junjiang Wang1,3, Gopika Punchhi1, Andrew Wolfe1,4, Sheng Wu1,4,5 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. Neuroscience Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion Qiao Li1, Cheng Qian1, Feng-Quan Zhou1,2 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. Developmental Biology Chemical Reversion of Conventional Human Pluripotent Stem Cells to a Naïve-like State with Improved Multilineage Differentiation Potency Tea Soon Park*1, Ludovic Zimmerlin*1, Rebecca Evans-Moses1, Elias T. Zambidis1 1Department of Oncology, Division of Pediatric Oncology and Institute for Cell Engineering, Johns Hopkins School of Medicine We present a protocol for efficient, bulk, and rapid chemical reversion of conventional lineage-primed human pluripotent stem cells (hPSC) into an epigenomically-stable naïve preimplantation epiblast-like pluripotent state. This method results in decreased lineage-primed gene expression and marked improvement in directed multilineage differentiation across a broad repertoire of conventional hPSC lines. Medicine Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRI—Application in Premanifest Huntington's Disease Dan Wu1, Andreia V. Faria1, Laurent Younes2,3,4, Christopher A. Ross5, Susumu Mori1,6, Michael I. Miller2,3,7 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. Biology Primary Cell Cultures from the Mouse Retinal Pigment Epithelium Peng Shang1, Nadezda A. Stepicheva1, Stacey Hose1, J. Samuel Zigler, Jr.2, Debasish Sinha1,2 1Department of Ophthalmology, University of Pittsburgh School of Medicine, 2Wilmer Eye Institute, The Johns Hopkins University School of Medicine The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE. Developmental Biology Long-term Behavioral and Reproductive Consequences of Embryonic Exposure to Low-dose Toxicants Mahlet D. Mersha1, Karla R. Sanchez2, Murali K. Temburni2, Harbinder S. Dhillon2 1Department of Neurology, Johns Hopkins School of Medicine, 2Department of Biological Sciences, Delaware State University Exposure to environmental toxicants can acutely impact development with long-term effects. Detailed protocols are provided to illustrate a strategy using an effective lab model to study the effect of early embryonic exposure to bisphenol A. We provide fecundity and behavioral assays to monitor the effectiveness of our toxicant exposure bio-assays. Medicine Posterior Semicircular Canal Approach for Inner Ear Gene Delivery in Neonatal Mouse Kevin Isgrig1, Wade W. Chien1,2 1National Institute on Deafness and Other Communication Disorders, National Institutes of Health, 2Department of Otolaryngology-Head & Neck Surgery, Johns Hopkins School of Medicine In this study, we describe the posterior semicircular canal approach as a reliable method for inner ear gene delivery in neonatal mice. We show that gene delivery through the posterior semicircular canal is able to perfuse the entire inner ear. Medicine Detecting Anastasis In Vivo by CaspaseTracker Biosensor Ho Man Tang1,2, Ming Chiu Fung2, Ho Lam Tang3 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. Cancer Research Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells Raffaella Spina1, Dillon M. Voss1, Laura Asnaghi2, Andrew Sloan1,3, Eli E. Bar1 1Department of Neurological Surgery, Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, 2Department of Pathology, Johns Hopkins University, School of Medicine, 3Department of Neurological Surgery, University Hospital-Case Medical Center, Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine An efficient screening protocol is presented for the identification of small molecules that promote astroglial differentiation in glioblastoma stem cells (GSCs). The assay is based on a stem cell differentiation reporter whereby the expression of the enhanced GFP (eGFP) is driven by the human GFAP promoter. Medicine Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique Benno Cardini*1, Rupert Oberhuber*1, Sven R Hein1, Rebecca Eiter1, Martin Hermann2, Markus Kofler1,3, Stefan Schneeberger1, Gerald Brandacher1,4, Manuel Maglione1 1Center of Operative Medicine, Department of Visceral, Transplant and Thoracic Surgery, Medical University Innsbruck, 2Department of Anesthesiology and Critical Care Medicine, Medical University Innsbruck, 4Department of Cardiac Surgery, Medical University Innsbruck, 3Department 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. Bioengineering Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy Lijuan He*1,2, Alexandra Sneider*1, Weitong Chen1, Michelle Karl1, Vishnu Prasath3, Pei-Hsun Wu1,2, Gunnar Mattson3, Denis Wirtz1,2,4 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. Biology Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation Yulia Artemenko1, Peter N. Devreotes2 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. Neuroscience A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila Mingkuan Sun1, Liam L. Chen1 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. Biochemistry Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding Chirag Vasavda*1, Nicholas W. Zaccor*1, Paul C. Scherer1, Charlotte J. Sumner1,2, Solomon H. Snyder1,3,4 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. Biology Flash-and-Freeze: A Novel Technique to Capture Membrane Dynamics with Electron Microscopy Shuo Li*1,2, Sumana Raychaudhuri*1, Shigeki Watanabe1,3 1Department of Cell Biology, Johns Hopkins School of Medicine, 2Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, 3Solomon H. Snyder Department of Neuroscience, Johns Hopkins School of Medicine We developed a novel technique in electron microscopy, "flash-and-freeze," that enables the visualization of membrane dynamics with ms temporal resolution. This technique combines the optogenetic stimulation of neurons with high-pressure freezing. Here, we demonstrate the procedures and describe the protocols in detail. Immunology and Infection Murine Full-thickness Skin Transplantation Chih-Hsien Cheng*1,2, Chen-Fang Lee*1,2, Madeline Fryer*3, Georg J. Furtmüller3, Byoungchol Oh3, Jonathan D. Powell1, Gerald Brandacher3 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. Behavior The Rodent Psychomotor Vigilance Test (rPVT): A Method for Assessing Neurobehavioral Performance in Rats and Mice Catherine M. Davis1, Peter G. Roma2, Robert D. Hienz1 1Division of Behavioral Biology, Department of Psychiatry and Behavioral Sciences, The Johns Hopkins University School of Medicine, 2Applied Behavior Biology Unit, Institutes for Behavior Resources A rat version of the human Psychomotor Vigilance Test (PVT) is described that measures aspects of attention similar to those measured with the human PVT, including aspects of human vigilance such as performance accuracy, motor speed, premature responding, and lapses in attention. Biochemistry Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling Sarah A. Head1, Jun O. Liu1,2 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. Neuroscience Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer Chun-Chieh Lin1, Olena Riabinina2, Christopher J. Potter1 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. Medicine A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure Matthew J. Hartsock1, Hongkwan Cho1, Lijuan Wu1, Wan-Ju Chen1, Junsong Gong1, Elia J. Duh1 1Department of Ophthalmology, School of Medicine, Johns Hopkins University This article describes a procedure for inducing retinal ischemia-reperfusion injury by elevated intraocular pressure in mice. Retinal ischemia-reperfusion injury by elevated intraocular pressure serves to model human pathologies characterized by compromised oxygen and nutrient delivery in the retina, enabling researchers to examine potential cellular mechanisms and treatments for human diseases of the retinal neurovascular unit. Medicine Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile Ling Li1, Klaus B. Piontek1, Vivek Kumbhari1, Masaharu Ishida2, Florin M. Selaru1 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. Developmental Biology Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation Lale Evsen1, Angelika Doetzlhofer1 1The Solomon H. Snyder Department of Neuroscience, The Center for Sensory Biology, The Johns Hopkins University, School of Medicine The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ. 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. Calabresi1, Emily G. Baxi1 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. Medicine Orthotopic Hind Limb Transplantation in the Mouse Georg J. Furtmüller*1, Byoungchol Oh*1, Johanna Grahammer*2, Cheng-Hung Lin3, Robert Sucher4, Madeline L. Fryer1, Giorgio Raimondi1, W.P. Andrew Lee1, Gerald Brandacher1 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). Medicine Adapted Resistance Training Improves Strength in Eight Weeks in Individuals with Multiple Sclerosis Jennifer L. Keller1, Nora Fritz1,2, Chen Chun Chiang1, Allen Jiang1, Tziporah Thompson3, Nicole Cornet1, Scott D. Newsome4, Peter A. Calabresi4, Kathleen Zackowski1,2,4 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. 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. Fryer1, Lawrence J. Gottlieb2,3, Michael R. Christy4, Gerald Brandacher1,5,6, Amir H. Dorafshar1,5,6 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. Bioengineering Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications Xiomara Calderón-Colón1, Giorgio Raimondi2, Jason J. Benkoski1, Julia B. Patrone3 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. Developmental Biology Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach M. Natalia Vergara1, Christian Gutierrez1, M. Valeria Canto-Soler1 1Wilmer Eye Institute, The Johns Hopkins University School of Medicine Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system. Developmental Biology Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche Peter Andersen1, Chulan Kwon1 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. Biology Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis Ho Lam Tang1,2,3, Ho Man Tang1,2,3, J. Marie Hardwick1, Ming Chiu Fung2 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. Biology Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity Nathachit Limjunyawong1, Jonathan Fallica1, Amritha Ramakrishnan2, Kausik Datta3, Matthew Gabrielson1, Maureen Horton1, Wayne Mitzner1 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. Biology Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method Saniya Fayzullina1, Lee J. Martin1 1Division of Neuropathology, Department of Pathology, Pathobiology Graduate Program, Johns Hopkins School of Medicine This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method for semi-automated analysis of TUNEL labeling. Medicine Combined In vivo Optical and µCT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice Nicholas M. Bernthal1, Brad N. Taylor2, Jeffrey A. Meganck2, Yu Wang3, Jonathan H. Shahbazian3, Jared A. Niska1, Kevin P. Francis2, Lloyd S. Miller3,4 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. Medicine Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique Rupert Oberhuber*1, Benno Cardini*1, Markus Kofler1, Paul Ritschl1, Robert Oellinger1, Felix Aigner1, Robert Sucher1, Stefan Schneeberger1, Johann Pratschke1, Gerald Brandacher2, Manuel Maglione1 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. Medicine A Preclinical Murine Model of Hepatic Metastases Kevin C. Soares1, Kelly Foley2, Kelly Olino1, Ashley Leubner2, Skye C. Mayo1, Ajay Jain1, Elizabeth Jaffee2, Richard D. Schulick3, Kiyoshi Yoshimura1,2, Barish Edil1,2, Lei Zheng1,2 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. 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. Kropp1, Sandra L. Chuppa1, Wai-Meng Kwok3, Joseph C. Wu2, Kenneth R. Boheler4,5, Rebekah L. Gundry1,6 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. Neuroscience Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology Hao Chang1, Yanshu Wang1, Hao Wu1, Jeremy Nathans1,2,3 1Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, 2Department of Neuroscience, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, 3Department of Ophthalmology, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine Mammalian skin contains a diverse array of structures - such as hair follicles and nerve endings - that exhibit distinctive patterns of spatial organization. Analyzing skin as a flat mount takes advantage of the 2-dimensional geometry of this tissue to produce full-thickness high-resolution images of skin structures. Medicine Fast and Accurate Exhaled Breath Ammonia Measurement Steven F. Solga1, Matthew L. Mudalel1, Lisa A. Spacek2, Terence H. Risby3 1 Ammonia is an important physiologic metabolite relevant to various disease and wellness states. It is also a difficult molecule to measure in breath, which demands particular precautions be taken to obtain accurate results. Not all factors influencing ammonia are known, but progress can be difficult without accounting for these factors. Biology Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR HoTae Lim1, In Young Choi1, Gabsang Lee1 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. Immunology and Infection Isolation of Double Negative αβ T Cells from the Kidney Maria N. Martina1, Samatha Bandapalle2, Hamid Rabb2, Abdel R. Hamad1 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. Biology Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus In Young Choi1, HoTae Lim1, Gabsang Lee1 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. Bioengineering Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides Yang Li1, Catherine A. Foss2, Martin G. Pomper2,3, S. Michael Yu1,3 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.
