Harvard Medical School 117 articles published in JoVE Neuroscience Using Home-based, Remotely Supervised, Transcranial Direct Current Stimulation for Phantom Limb Pain Kevin Pacheco-Barrios*1,2, Daniela Martinez-Magallanes*1, Cristina Xicota Naqui1,3, Marianna Daibes1, Elly Pichardo1, Alejandra Cardenas-Rojas1, David Crandell4, Anahita Dua5, Abhishek Datta6,7, Wolnei Caumo8,9,10, Felipe Fregni1 1Neuromodulation Center and Center for Clinical Research Learning, Spaulding Rehabilitation Hospital, Massachusetts General Hospital, Harvard Medical School, 2Vicerrectorado de Investigación, Unidad de Investigación para la Generación y Síntesis de Evidencias en Salud, Universidad San Ignacio de Loyola, 3Nursing Department, Universitat Internacional de Catalunya, 4Spaulding Rehabilitation Hospital, Harvard Medical School, 5Division of Vascular and Endovascular Surgery, Massachusetts General Hospital, Harvard Medical School, 6Research and Development, Soterix Medical, 7City College of New York, 8Post-Graduate Program in Medical Sciences, School of Medicine, Universidade Federal do Rio Grande do Sul (UFRGS), 9Laboratory of Pain & Neuromodulation, Hospital de Clinicas de Porto Alegre (HCPA), 10Pain and Palliative Care Service, Hospital de Clinicas de Porto Alegre (HCPA) The goal of this study is to describe a protocol for the home-based delivery of remotely supervised transcranial direct current stimulation (RS-tDCS) conserving the standard procedures of in-clinic practice, including safety, reproducibility, and tolerability. The participants included will be patients with phantom limb pain (PLP). Immunology and Infection Epithelial Cell Infection Analyses with Shigella Kender Poore*1, Bryan R. Lenneman*1,2, Christina S. Faherty1,2 1Mucosal Immunology and Biology Research Center, Division of Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital, 2Department of Pediatrics, Harvard Medical School The present protocol describes infection assays to interrogate Shigella adherence, invasion, and intracellular replication using in vitro epithelial cell lines. Neuroscience Manual Segmentation of the Human Choroid Plexus Using Brain MRI Deepthi Bannai*1,2, Yuan Cao*3,4, Matcheri Keshavan1,2, Martin Reuter5,6,7, Paulo Lizano1,2,8 1Department of Psychiatry, Beth Israel Deaconess Medical Center, 2Division of Translational Neuroscience, Beth Israel Deaconess Medical Center, 3Huaxi MR Research Center (HMRRC), Department of Radiology, West China Hospital of Sichuan University, 4Department of Psychiatry and Psychotherapy, Jena University Hospital, 5AI in Medical Imaging, German Center for Neurodegenerative Diseases (DZNE), 6A. A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital, 7Department of Radiology, Harvard Medical School, 8Department of Psychiatry, Harvard Medical School Despite the crucial role of the choroid plexus in the brain, neuroimaging studies of this structure are scarce due to the lack of reliable automated segmentation tools. The present protocol aims to ensure gold-standard manual segmentation of the choroid plexus that can inform future neuroimaging studies. Medicine Model of Ischemia and Reperfusion Injury in Rabbits Victor S. Alemany*1, Dominic P. Recco*1, Sitaram M. Emani1, Pedro J. del Nido1, James D. McCully1 1Department of Cardiac Surgery, Boston Children’s Hospital, Harvard Medical School The present study demonstrates a highly reproducible animal model of acute regional myocardial ischemia and reperfusion injury in rabbits using a left mini-thoracotomy for survival cases or a midline sternotomy for non-survival cases. Biology In Vitro Method to Study Sex-Based Differences in Conjunctival Goblet Cells Jeffrey A. Bair1, Darlene A. Dartt1, Menglu Yang1 1Department of Ophthalmology Schepens Eye Research Institute, Massachusetts Eye and Ear, Harvard Medical School Phenol red-free/fetal bovine serum-free medium is a better option than advanced RPMI to eliminate exogenous hormones without altering the normal function of conjunctival goblet cells in the study of sex-based differences. Medicine A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition Gregor Gierlinger1, Lavinia Rech1, Sitaram M. Emani1, Pedro J. del Nido1, Ingeborg Friehs1 1Department of Cardiac Surgery, Boston Children’s Hospital, Harvard Medical School This work presents an animal model of endothelial-to-mesenchymal transition-induced fibrosis, as seen in congenital cardiac defects such as critical aortic stenosis or hypoplastic left heart syndrome, which allows for detailed histological tissue evaluation, the identification of regulatory signaling pathways, and the testing of treatment options. Medicine Modified Tail Vein and Penile Vein Puncture for Blood Sampling in the Rat Model Laura Charlès1,2,3, Thomas Agius*4,5, Irina Filz von Reiterdank*1,2,3,6, Janna Hagedorn1,2,3, Yanis Berkane1,2,3,7, Hyshem H. Lancia1,2,3, Basak E. Uygun2,3,8, Korkut Uygun2,3,8, Curtis L. Cetrulo Jr.1,2,3,9, Mark A. Randolph1,2,3,9, Alexandre G. Lellouch1,2,3,8 1Vascularized Composite Allotransplantation Laboratory, Massachusetts General Hospital, 2Harvard Medical School, 3 Here, we present a protocol to offer rapid, easy, and reliable blood collection alternatives for the rat model. We describe three different blood sampling methods according to the context: tail vein puncture under anesthesia or on a conscious animal, and dorsal penile vein puncture under anesthesia. Biology Monitoring On-Target Signaling Responses in Larval Zebrafish - Z-REX Unmasks Precise Mechanisms of Electrophilic Drugs and Metabolites Kuan-Ting Huang1, Phillippe Ly1, Jesse R. Poganik2, Saba Parvez3, Marcus J. C. Long4, Yimon Aye1 1Swiss Federal Institute of Technology Lausanne (EPFL), 2Division of Genetics, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, 3Department of Pharmacology and Toxicology, College of Pharmacy, University of Utah, 4University of Lausanne (UNIL) Zebrafish targeting reactive electrophiles and oxidants (Z-REX) is a chemical biology-based method for the investigation of reactive small-molecule signaling. This technique can be applied to live fish of different developmental stages. Here, we couple standard assays in zebrafish with Z-REX for signaling pathway analysis. Biology Effective Rapid Blood Perfusion in Xenopus Rachael A. Jonas-Closs1, Leonid Peshkin1 1Department of Systems Biology, Harvard Medical School Presented here is an effective rapid blood perfusion protocol to prepare tissue samples from African clawed frogs for transcriptomics and proteomics studies. Neuroscience Robust Tissue Fabrication for Long-Term Culture of iPSC-Derived Brain Organoids for Aging Research Lena Sophie Koch1, David Choy Buentello2,3, Kerensa Broersen1 1Department of Applied Stem Cell Technologies, TechMed Centre Enschede, University of Twente, 2Centro de Biotecnología-FEMSA, Tecnologico de Monterrey, 3Department of Genetics, Harvard Medical School The present protocol provides a step-by-step procedure for the reproducible generation, maintenance, and aging of cerebral organoids derived from human-induced pluripotent stem cells (iPSCs). This method enables culturing and maturing cerebral organoids for extended periods, which facilitates the modeling of processes involved in brain aging and age-related pathogenesis. Biology Generation of Brown Fat-Specific Knockout Mice Using a Combined Cre-LoxP, CRISPR-Cas9, and Adeno-Associated Virus Single-Guide RNA System Tadataka Tsuji1, Yang Zhang1, Yu-Hua Tseng1 1Section on Integrative Physiology and Metabolism, Research Division, Joslin Diabetes Center, Harvard Medical School In this protocol, we describe the technical procedures to generate brown adipose tissue (BAT)-specific knockout mice leveraging a combined Cre-LoxP, CRISPR-Cas9, and adeno-associated virus (AAV) single-guide RNA (sgRNA) system. The described steps include the design of the sgRNAs, the preparation of the AAV-sgRNA particles, and the microinjection of AAV into the BAT lobes. Immunology and Infection A Novel Method to Determine the Longitudinal Antibacterial Activity of Drug-Eluting Materials Amita Sekar1,2, Sashank Lekkala1, Ebru Oral1,2 1Harris Orthopaedics Laboratory, Massachusetts General Hospital, 2Department of Orthopaedic Surgery, Harvard Medical School Here, we present a protocol to evaluate the antibacterial efficacy of an antibiotic-eluting polymer to simulate prophylactic clinical application by using a commercially available real-time ATP-based luminescent microbial viability assay. This method enables the monitoring of the longitudinal activity of drug-eluting materials and can be widely adapted to test anti-microbial drug delivery platforms. Biology Real-Time Analysis of Bioenergetics in Primary Human Retinal Pigment Epithelial Cells Using High-Resolution Respirometry Tessa C. Fitch*1,2, Scott I. Frank*1, Yutong Kelly Li1, Magali Saint-Geniez1,2, Leo A. Kim1,2, Daisy Y. Shu1,2 1Schepens Eye Research Institute of Mass. Eye and Ear, 2Department of Ophthalmology, Harvard Medical School The metabolic status of human retinal pigment epithelial cells (H-RPE) reflects their health and function. Presented here is an optimized protocol for examining the real-time metabolic flux of H-RPE using high-resolution respirometry. Immunology and Infection The Superficial Inferior Epigastric Artery Axial Flap to Study Ischemic Preconditioning Effects in a Rat Model Yanis Berkane*1,2,3,5, Austin Alana Shamlou*1,2,4,5, Jose Reyes1,2, Hyshem H. Lancia1,2,5, Irina Filz von Reiterdank1,2,5,6,7, Nicolas Bertheuil3, Basak E. Uygun2,5,6, Korkut Uygun1,2,5,6, William G. Austen Jr.2,4, Curtis L. Cetrulo Jr.1,2,4,5, Mark A. Randolph1,2,4,5, Alexandre G. Lellouch1,2,5,6,8 1Vascularized Composite Allotransplantation Laboratory, Massachusetts General Hospital, 2Harvard Medical School, 3Department of Plastic, Reconstructive and Aesthetic Surgery, Rennes University Hospital Center (CHU de Rennes), University of Rennes 1, 4Plastic Surgery Research Laboratory, Massachusetts General Hospital, 5 This protocol describes harvesting, suturing, and monitoring fasciocutaneous flaps in rats that allow for good visualization and manipulation of blood flow through the superficial inferior epigastric vessels by means of clamping and ligating the femoral vessels. This is critical for studies involving ischemic preconditioning. Biochemistry Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells Sarah-Luisa Dülk1, Silvia Rouskin1 1Department of Microbiology, Harvard Medical School The protocol provides instruction for modifying RNA with dimethyl sulfate for mutational profiling experiments. It includes in vitro and in vivo probing with two alternative library preparation methods. Biology Bioluminescent Monitoring of Graft Survival in an Adoptive Transfer Model of Autoimmune Diabetes in Mice Taylor Stewart1, Kevin Bode2, Stephan Kissler2, Peng Yi1 1Section for Islet Cell and Regenerative Biology, Joslin Diabetes Center, Harvard Medical School, 2Section for Immunology, Joslin Diabetes Center, Harvard Medical School This protocol describes a straightforward and minimally invasive method for transplanting and imaging NIT-1 cells in non-obese diabetic (NOD)-severe combined immunodeficient mice challenged with splenocytes purified from spontaneously diabetic NOD mice. Medicine Investigation of the Electrophysiological and Thermographic Safety Parameters of Surgical Energy Devices During Thyroid and Parathyroid Surgery in a Porcine Model Hsin-Yi Tseng1, Tzu-Yen Huang1, Jia Joanna Wang1,2, Yi-Chu Lin1, I-Cheng Lu3, Feng-Yu Chiang4, Gianlorenzo Dionigi5,6, Gregory W. Randolph7, Che-Wei Wu1,8 1Department of Otorhinolaryngology-Head and Neck Surgery, International Thyroid Surgery Center, Kaohsiung Medical University Hospital, Kaohsiung Medical University, 2Department of Otolaryngology-Head and Neck Surgery, Kaohsiung Municipal Siaogang Hospital, Kaohsiung Medical University, 3Department of Anesthesiology, Kaohsiung Municipal Siaogang Hospital, Kaohsiung Medical University, 4Department of Otolaryngology-Head and Neck Surgery, E-Da Hospital, I-Shou University, 5Division of General Surgery, Endocrine Surgery Section, Istituto Auxologico Italiano (IRCCS), 6Department of Pathophysiology and Transplantation, Faculty of Medicine and Surgery, University of Milan, 7Division of Thyroid and Parathyroid Endocrine Surgery, Department of Otolaryngology-Head and Neck Surgery, Massachusetts Eye and Ear Infirmary, Harvard Medical School, 8Center for Liquid Biopsy and Cohort Research, and Faculty of Medicine, College of Medicine, Kaohsiung Medical University The safe application of newly developed surgical energy devices in thyroid/parathyroid surgery attracts the attention of surgeons. Animal experimental models can avoid unnecessary trials and errors in human surgery. This report aims to demonstrate electrophysiological and thermographic methods to evaluate the safety parameters of SEDs in thyroid/parathyroid surgery. Neuroscience Measuring Contralateral Silent Period Induced by Single-Pulse Transcranial Magnetic Stimulation to Investigate M1 Corticospinal Inhibition Ingrid Rebello-Sanchez1, Joao Parente1, Kevin Pacheco-Barrios1,2, Anna Marduy1, Danielle Carolina Pimenta1, Daniel Lima1, Eric Slawka1, Alejandra Cardenas-Rojas1, Gleysson Rodrigues Rosa1, Kamran Nazim3, Abhishek Datta3,4, Felipe Fregni1 1Neuromodulation Center and Center for Clinical Research Learning, Spaulding Rehabilitation Hospital and Massachusetts General Hospital, Harvard Medical School, 2Universidad de Investigación para la Generación y Síntesis de Evidencia en Salud, Universidad San Ignacio de Loyola, 3Research and Development, Soterix Medical, 4City College of New York Contralateral silent period (cSP) assessment is a promising biomarker to index cortical excitability and treatment response. We demonstrate a protocol to assess cSP intended for studying M1 corticospinal inhibition of upper and lower limbs. Medicine Intraoperative Ultrasound in Spinal Surgery Melissa M.J. Chua1, Viren S. Vasudeva1, Yi Lu1 1Brigham and Women's Hospital, Harvard Medical School Here, we present a protocol on the use of intraoperative ultrasound in spinal surgery, particularly in cases of intradural lesions and lesions in the ventral spinal canal when using a posterior approach. Medicine Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability Kiran Bora*1, Zhongxiao Wang*1, Felix Yemanyi1, Meenakshi Maurya1, Alexandra K. Blomfield1, Yohei Tomita1, Jing Chen1 1Department of Ophthalmology, Boston Children’s Hospital, Harvard Medical School This protocol illustrates an in vitro endothelial cell transcytosis assay as a model to evaluate inner blood-retinal barrier permeability by measuring the ability of human retinal microvascular endothelial cells to transport horseradish peroxidase across cells in caveolae-mediated transcellular transport processes. Neuroscience Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain Gabriel S. Jensen1,2, Jake W. Willows2, David T. Breault3, Kristy L. Townsend1,2 1Graduate School of Biomedical Science and Engineering, University of Maine, 2Department of Neurosurgery, Ohio State University Wexner Medical Center, 3Boston Children’s Hospital, Harvard Medical School The ability to permanently mark stem cells and their progeny with a fluorophore using an inducible transgenic lineage tracing mouse line allows for spatial and temporal analysis of activation, proliferation, migration, and/or differentiation in vivo. Lineage tracing can reveal novel information about lineage commitment, response to intervention(s), and multipotency. Bioengineering Fabricating Highly Open Porous Microspheres (HOPMs) via Microfluidic Technology Sheng-Chang Luo1,2, Ying Wang3, Ranjith Kumar Kankala1,2, Yu Shrike Zhang4, Ai-Zheng Chen1,2 1Institute of Biomaterials and Tissue Engineering, Huaqiao University, 2Fujian Provincial Key Laboratory of Biochemical Technology, Huaqiao University, 3Affiliated Dongguan Hospital, Southern Medical University, 4Division of Engineering in Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School The present protocol describes the fabrication of poly(lactic-co-glycolic acid)-based highly open porous microspheres (HOPMs) via the single-emulsion formulation based facile microfluidic technology. These microspheres have potential applications in tissue engineering and drug screening. Developmental Biology A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo Wei Zhang*1, Xinjuan Lei*1, Xin Zhou*2,3, Boling He1, Liqin Xiao1, Huimin Yue1, Shulin Wang1, Yuting Sun1, Yajun Wu1, Liyang Wang1,4, George Ghartey-Kwansah1, Odell D. Jones5, Joseph L. Bryant6, MengMeng Xu7, Jianjie Ma3, Xuehon Xu1 1National Engineering Laboratory for Resource Development of Endangered Crude Drugs in Northwest of China/CGDB, Shaanxi Normal University College of Life Sciences, 2 Here, we describe a protocol for detection and localization of Drosophila embryo protein and RNA from collection to pre-embedding and embedding, immunostaining, and mRNA in situ hybridization. Biochemistry Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy Nandini Mani*1,2, Michelle F. Marchan*1, Radhika Subramanian1,2 1Molecular Biology, Massachusetts General Hospital, 2Department of Genetics, Harvard Medical School Here, a TIRF microscopy-based in vitro reconstitution assay is presented to simultaneously quantify and compare the dynamics of two microtubule populations. A method is described to simultaneously view the collective activity of multiple microtubule-associated proteins on crosslinked microtubule bundles and single microtubules. Medicine Chemiluminescence-based Assays for Detection of Nitric Oxide and its Derivatives from Autoxidation and Nitrosated Compounds Raffaele Di Fenza1,3, Binglan Yu1,3, Ryan W. Carroll2,3, Lorenzo Berra1,3 1Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, 2Department of Pediatrics, Massachusetts General Hospital, 3Harvard Medical School Here, we present protocols for detecting nitric oxide and its biologically relevant derivatives using chemiluminescence-based assays with high sensitivity. Neuroscience Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice Krutika Joshi1, Alana Kirby1, Jianguo Niu1, Veronique VanderHorst1 1Beth Israel Deaconess Medical Center, Harvard Medical School Stereotaxic surgery to target brain sites in mice commonly involves access through the skull bones and is guided by skull landmarks. Here we outline an alternative stereotaxic approach to target the caudal brainstem and upper cervical spinal cord via the cisterna magna that relies on direct visualization of brainstem landmarks. Biology Isolation of Human Neutrophils from Whole Blood and Buffy Coats Alan Y. Hsu1, Zhicheng Peng1, Hongbo Luo1, Fabien Loison1,2 1Department of Pathology, Harvard Medical School, Department of Lab Medicine, Children’s Hospital Boston, and Dana-Farber/Harvard Cancer Center, 2Department of Microbiology, Faculty of Science, Mahidol University This protocol details a method for the isolation of neutrophils from whole blood, buffy coats, or leukapheresis membranes, achieving good yield, high purity, and minimal cell activation. We utilize gradient purification, red blood cell (RBC) sedimentation, and RBC lysis to obtain a high-quality/purity neutrophil preparation. Immunology and Infection Conjunctival Commensal Isolation and Identification in Mice Kirsten Smith-Page1, Abirami Kugadas1, Tiffany Lin1, Mary Delaney2,3, Lynn Bry2,3, Mihaela Gadjeva1 1Department of Medicine, Division of Infectious Diseases, Brigham and Women's Hospital, Harvard Medical School, 2Massachusetts Host-Microbiome Center, Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, 3Clinical Microbiology Laboratory, Department of Pathology, Brigham and Women's Hospital, Harvard Medical School Presented here is a protocol for the isolation and amplification of aerobic and facultative anaerobic mouse conjunctival commensal bacteria using a unique eye swab and culture-based enrichment step with subsequent identification by microbiological based methods and MALDI-TOF mass spectrometry. Biology Partial Hepatectomy in Adult Zebrafish Isaac M. Oderberg1,2, Wolfram Goessling1,2,3 1Harvard Medical School, 2 This protocol describes the procedure for removing the ventral lobe of the liver in adult zebrafish to enable the study of liver regeneration. Biology Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells Blanca Chinchilla1, Heran Getachew1, Rosario Fernandez-Godino1 1Ocular Genomics Institute of Massachusetts Eye and Ear, Harvard Medical School This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours. Medicine A Large Animal Model for Acute Kidney Injury by Temporary Bilateral Renal Artery Occlusion Ilias P. Doulamis1, Alvise Guariento1, Mossab Y. Saeed1, Rio S. Nomoto1, Thomas Duignan1, Pedro J. del Nido1, James D. McCully1 1Department of Cardiac Surgery, Boston Children’s Hospital, Harvard Medical School This study presents a highly reproducible large animal model of renal ischemia-reperfusion injury in swine using temporary percutaneous bilateral balloon-catheter occlusion of the renal arteries for 60 min and reperfusion for 24 h. Neuroscience Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells Sovannarath Pong1,2,3, Paulo Lizano1,2,3,4, Rakesh Karmacharya1,3,4,5 1Center for Genomic Medicine, Massachusetts General Hospital, 2Department of Psychiatry, Beth Israel Deaconess Medical Center, 3Chemical Biology and Therapeutic Science Program, Broad Institute of MIT and Harvard, 4Department of Psychiatry, Harvard Medical School, 5Schizophrenia and Bipolar Disorder Program, McLean Hospital This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model. Biochemistry A Model Membrane Platform for Reconstituting Mitochondrial Membrane Dynamics Yifan Ge1, Sivakumar Boopathy1, Adam Smith2, Luke H. Chao1,3 1Department of Molecular Biology, Massachusetts General Hospital, 2Department of Chemistry, University of Akron, 3Department of Genetics, Harvard Medical School Mitochondrial fusion is an important homeostatic reaction underlying mitochondrial dynamics. Described here is an in vitro reconstitution system to study mitochondrial inner-membrane fusion that can resolve membrane tethering, docking, hemifusion, and pore opening. The versatility of this approach in exploring cell membrane systems is discussed. Biology An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis Yohei Tomita1, Zhuo Shao2, Bertan Cakir1, Yumi Kotoda1, Zhongjie Fu1,3, Lois E.H. Smith1 1Department of Ophthalmology, Boston Children's Hospital, Harvard Medical School, 2Department of Clinical and Metabolic Genetics, Hospital for Sick Children, University of Toronto, 3Manton Center for Orphan Disease, Harvard Medical School, Boston Children's Hospital This protocol presents choroid sprouting assay, an ex vivo model of microvascular proliferation. This assay can be used to assess pathways involved in proliferating choroidal micro vessels and assess drug treatments using wild type and genetically modified mouse tissue. Biology A Single Cell Dissociation Approach for Molecular Analysis of Urinary Bladder in the Mouse Following Spinal Cord Injury Hussein Atta*1,2, Ali Hashemi Gheinani*1,2, Amanda Wacker1, Yaser Heshmati3,4,5, Alex Bigger-Allen1,6, George Lambrinos1,2, Yao Gao2,7, Diane R. Bielenberg2,7, Rosalyn M. Adam1,2 1 The goal of this protocol is to apply an optimized tissue dissociation protocol to a mouse model of spinal cord injury and validate the approach for single cell analysis by flow cytometry. Medicine Using an Automated Hirschberg Test App to Evaluate Ocular Alignment Gang Luo1, Shrinivas Pundlik1, Matteo Tomasi2, Kevin Houston1 1Schepens Eye Research Institute, Mass Eye & Ear, Harvard Medical School, 2EyeNexo, LLC We present a protocol of using a smartphone app to perform Hirschberg test for measuring manifest and intermittent ocular misalignment (strabismus) under near and far fixation conditions. Bioengineering Bioinspired Soft Robot with Incorporated Microelectrodes Ting Wang1,2, Bianca Migliori1,3, Beatrice Miccoli1,4, Su Ryon Shin1 1Division of Engineering in Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 2School of Medicine, Jiangsu University, 3Tech4Health and Neuroscience Institutes, NYU Langone Health, 4Department of Electronics and Telecommunication, Politecnico di Torino A bioinspired scaffold is fabricated by a soft photolithography technique using mechanically robust and electrically conductive hydrogels. The micropatterned hydrogels provide directional cardiomyocyte cell alignment, resulting in a tailored direction of actuation. Flexible microelectrodes are also integrated into the scaffold to bring electrical controllability for a self-actuating cardiac tissue. Editorial Little Fish, Big Questions: A Collection of Modern Techniques for Mexican Tetra Research Misty R. Riddle1, Clifford J. Tabin1 1Genetics Department, Blavatnik Institute, Harvard Medical School Neuroscience Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons Debkanya Datta1,2, Anju Vasudevan1,2 1Department of Psychiatry, Harvard Medical School, 2Angiogenesis and Brain Development Laboratory, Division of Basic Neuroscience, McLean Hospital We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons. Neuroscience Functional MRI in Conjunction with a Novel MRI-compatible Hand-induced Robotic Device to Evaluate Rehabilitation of Individuals Recovering from Hand Grip Deficits Mark P. Ottensmeyer1,2, Shasha Li2,3,4, Gianluca De Novi1,2, A. Aria Tzika2,3,4 1Medical Device & Simulation Laboratory, Department of Radiology, Massachusetts General Hospital, 2Harvard Medical School, 3NMR Surgical Laboratory, Department of Surgery, Center for Surgery, Innovation and Bioengineering, Massachusetts General Hospital, Harvard Medical School, 4Athinoula A. Martinos Center of Biomedical Imaging, Department of Radiology, Massachusetts General Hospital, Harvard Medical School We performed functional MRI using a novel MRI-compatible hand-induced robotic device to evaluate its utility for monitoring hand motor function in individuals recovering from neurological deficits. Medicine Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods Francisco J. Molina Estevez*1, Tyler D. Mathews*1, Alessandra Biffi1,2, Marco Peviani1,2 1 We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles. Neuroscience Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice Ryosuke Fujiki1,2,3,4,9, Joun Y. Lee1,2,10, Julie A. Jurgens1,2,3,7, Mary C. Whitman2,5,6, Elizabeth C. Engle1,2,3,4,5,6,7,8 1 This work presents a protocol to yield homogeneous cell cultures of primary oculomotor, trochlear, and spinal motor neurons. These cultures can be used for comparative analyses of the morphological, cellular, molecular, and electrophysiological characteristics of ocular and spinal motor neurons. Immunology and Infection Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice Xiaofang Xiong1, Fengyang Lei2, Mohammad Haque1, Jianxun Song1 1Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, 2Department of Ophthalmology, Harvard Medical School Presented here is a protocol for the effective suppression of hepatitis B virus (HBV) replication in mice by utilizing adoptive cell transfer (ACT) of stem cell-derived viral antigen (Ag)-specific T lymphocytes. This procedure may be adapted for potential ACT-based immunotherapy of HBV infection. Biochemistry Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation Emily A. Brown1,2, Steven C. Neier3,4, Claudia Neuhauser5, Adam G. Schrum6,7,8, Stephen E.P. Smith1,2,9 1 Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network. Bioengineering Implementation of Interference Reflection Microscopy for Label-free, High-speed Imaging of Microtubules Mohammed Mahamdeh1,2, Jonathon Howard1 1Department of Molecular Biophysics and Biochemistry, Yale University, 2Harvard Medical School, Harvard University This protocol is a guide for implementing interference reflection microscopy on a standard fluorescence microscope for label-free, high-contrast, high-speed imaging of microtubules using in vitro surfaces assays. Neuroscience Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth Mary C. Whitman1,2,3, Jessica L. Bell1,3, Elaine H. Nguyen1,3, Elizabeth C. Engle1,2,3,4,5,6 1 An ex vivo slice assay allows oculomotor nerve outgrowth to be imaged in real time. Slices are generated by embedding E10.5 IslMN:GFP embryos in agarose, slicing on a vibratome, and growing in a stage-top incubator. The role of axon guidance pathways is assessed by adding inhibitors to the culture media. Immunology and Infection Isolation and Quantitative Evaluation of Brush Cells from Mouse Tracheas Saltanat Ualiyeva1, Eri Yoshimoto1, Nora A. Barrett1, Lora G. Bankova1 1Division of Rheumatology, Allergy and Immunology, Brigham and Women's Hospital, Harvard Medical School Brush cells are rare cholinergic chemosensory epithelial cells found in the naïve mouse trachea. Due to their limited numbers, ex vivo evaluation of their functional role in airway immunity and remodeling is challenging. We describe a method for isolation of tracheal brush cells by flow cytometry. Cancer Research Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland Dongxi Xiang1,2, Luwei Tao1,2, Zhe Li1,2 1 The goal of this protocol is to describe a new breast cancer modeling approach based on the intraductal injection of Cre-expressing adenovirus into mouse mammary glands. This approach allows both cell-type- and organ-specific manipulation of oncogenic events in a temporally controlled manner. Medicine Assessment and Characterization of Hyaloid Vessels in Mice Zhongxiao Wang1, Chi-Hsiu Liu1, Shuo Huang1, Jing Chen1,2 1Department of Ophthalmology, Boston Children's Hospital, Harvard Medical School, 2The Manton Center for Orphan Disease Research, Boston Children's Hospital, Harvard Medical School This protocol describes both in vivo and ex vivo methods to fully visualize and characterize hyaloid vessels, a model of vascular regression in mouse eyes, using optical coherence tomography and fundus fluorescein angiography for the live imaging and ex vivo isolation and subsequent flat mount of hyaloid for quantitative analysis. Developmental Biology Surgical Size Reduction of Zebrafish for the Study of Embryonic Pattern Scaling Kana Ishimatsu1, Anna Cha1, Zach M. Collins1, Sean G. Megason1 1Department of Systems Biology, Harvard Medical School Here, we describe a method for reducing the size of zebrafish embryos without disrupting normal developmental processes. This technique enables the study of pattern scaling and developmental robustness against size change. Neuroscience Real-time Video Projection in an MRI for Characterization of Neural Correlates Associated with Mirror Therapy for Phantom Limb Pain Faddi G. Saleh Velez1,2, Camila B. Pinto1,3, Emma S. Bailin4, Marionna Münger1, Andrew Ellison5, Beatriz T. Costa1, David Crandell6, Nadia Bolognini7,8, Lotfi B. Merabet4, Felipe Fregni1 1Laboratory of Neuromodulation & Center for Clinical Research Learning, Department of Physical Medicine and Rehabilitation, Harvard Medical School, Spaulding Rehabilitation Hospital, 2University of Chicago Medical Center, Department of Neurology, University of Chicago, 3Department of Neuroscience and Behavior, Psychology Institute, University of Sao Paulo, 4The Laboratory for Visual Neuroplasticity, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, 5Center for Biomedical Imaging, Department of Anatomy and Neurobiology, Boston University School of Medicine, 6Spaulding Rehabilitation Hospital, Harvard Medical School, 7Department of Psychology & Milan Center for Neuroscience, University of Milano-Bicocca, 8Neuropsychological Laboratory, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Istituto Auxologico Italiano We present a novel combined behavioral and neuroimaging protocol employing real-time video projection for the purpose of characterizing the neural correlates associated with mirror therapy within the magnetic resonance imaging scanner environment in leg amputee subjects with phantom limb pain. Immunology and Infection Antimicrobial Synergy Testing by the Inkjet Printer-assisted Automated Checkerboard Array and the Manual Time-kill Method Thea Brennan-Krohn1,2,3, James E Kirby1,3 1Department of Pathology, Beth Israel Deaconess Medical Center, 2 Antimicrobial synergy testing is used to evaluate the effect of two or more antibiotics used in combination and is typically performed by one of two methods: the checkerboard array or the time-kill assay. Here, we present an automated, inkjet printer-assisted checkerboard array synergy technique and a classic time-kill synergy study. Bioengineering Cell-free Protein Expression Using the Rapidly Growing Bacterium Vibrio natriegens Daniel J. Wiegand1,2, Henry H. Lee1, Nili Ostrov*1, George M. Church*1,2 1Department of Genetics, Harvard Medical School, 2Wyss Institute for Biologically Inspired Engineering Cell-free expression systems are powerful and cost-efficient tools for the high-throughput synthesis and screening of important proteins. Here, we describe the preparation of cell-free protein expression system using Vibrio natriegens for the rapid protein production using plasmid DNA, linear DNA, and mRNA template. Neuroscience In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films Skyler L. Jackman1, Christopher H. Chen2, Wade G. Regehr2 1Vollum Institute, Oregon Health and Science University, 2Harvard Medical School Here, we present a method for delivering viral expression vectors into the brain using silk fibroin films. This method allows targeted delivery of expression vectors using silk/AAV coated optical fibers, tapered optical fibers, and cranial windows. Genetics Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry Misty Riddle1, Brian Martineau1, Megan Peavey1, Clifford Tabin1 1Harvard Medical School In this protocol, we demonstrate how to breed Astyanax mexicanus adults, raise the larvae, and perform whole-mount immunohistochemistry on post-larval fish to compare the phenotypes of surface and cave morphotypes. Cancer Research Repression of Multiple Myeloma Cell Growth In Vivo by Single-wall Carbon Nanotube (SWCNT)-delivered MALAT1 Antisense Oligos Jianhong Lin*1,2, Yi Hu*1, Jian-Jun Zhao1 1Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, 2Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School This manuscript describes the synthesis of a single-wall carbon nanotube (SWCNT)-conjugated MALAT1 antisense gapmer DNA oligonucleotide (SWCNT-anti-MALAT1), which demonstrates the reliable delivery of the SWCNT and the potent therapeutic effect of anti-MALAT1 in vitro and in vivo. Methods used for synthesis, modification, conjugation, and injection of SWCNT-anti-MALAT1 are described. Cancer Research Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer's Patches Yavuz F. Yazicioglu1,2, Halil I. Aksoylar1,2, Rinku Pal1,2, Nikolaos Patsoukis1,2, Vassiliki A. Boussiotis1,2 1Division of Hematology-Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, 2Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School In this study, we present a novel and effective protocol for the isolation of lymphocytes from Peyer's Patches (PPs), which can be subsequently used for in vivo and in vitro functional assays as well as flow cytometric studies of follicular T helper and germinal center B cells. Neuroscience A Protocol for Transcranial Photobiomodulation Therapy in Mice Farzad Salehpour1,2, Luis De Taboada3, Paolo Cassano4,5,6, Farzin Kamari1, Javad Mahmoudi1, Sohrab Ahmadi-Kandjani7, Seyed Hossein Rasta8,9,10, Saeed Sadigh-Eteghad1 1Neurosciences Research Center, Tabriz University of Medical Sciences, 2ProNeuroLIGHT LLC, 3LiteCure LLC, 4Department of Psychiatry, Harvard Medical School, 5Depression Clinical and Research Program, Department of Psychiatry, Massachusetts General Hospital, 6Center for Anxiety and Traumatic Stress Disorders, Department of Psychiatry, Massachusetts General Hospital, 7Research Institute for Applied Physics and Astronomy, University of Tabriz, 8Department of Medical Physics, Tabriz University of Medical Sciences, 9Department of Medical Bioengineering, Tabriz University of Medical Sciences, 10School of Medical Sciences, University of Aberdeen Photobiomodulation therapy is an innovative noninvasive modality for the treatment of a wide range of neurological and psychiatric disorders and can also improve healthy brain function. This protocol includes a step-by-step guide to performing brain photobiomodulation in mice by transcranial light delivery, which can be adapted for use in other laboratory rodents. Bioengineering Apparatus for Harvesting Tissue Microcolumns Joshua Tam1,2, William Farinelli1, Walfre Franco1,2, R. Rox Anderson1,2 1Wellman Center for Photomedicine, Massachusetts General Hospital, 2Department of Dermatology, Harvard Medical School Here we describe a protocol for producing harvesting needles that can be used to collect full-thickness skin tissue without causing donor site scarring. The needles can be combined with a simple collection system to achieve high-volume harvesting. Immunology and Infection Listeria monocytogenes Infection of the Brain Pallab Ghosh1, Darrren E. Higgins1 1Department of Microbiology and Immunobiology, Harvard Medical School During infection, Listeria monocytogenes is capable of crossing the blood-brain barrier to colonize the brain. In this protocol, we demonstrate how to assess bacterial colonization of organs following infection of mice. A procedure to perform whole organ perfusion for specific determination of bacterial numbers in the brain parenchyma is provided. Genetics CRISPR Guide RNA Cloning for Mammalian Systems Sathiji Nageshwaran*1,2, Alejandro Chavez*1,2,3, Nan Cher Yeo1,2, Xiaoge Guo1,2, Alissa Lance-Byrne1, Angela Tung1, James J. Collins1,4,5,6,7, George M. Church1,2 1Wyss Institute for Biologically Inspired Engineering, Harvard University, 2Department of Genetics, Harvard Medical School, 3Department of Pathology, Massachusetts General Hospital, 4Institute for Medical Engineering & Science, Massachusetts Institute of Technology, 5Synthetic Biology Center, Massachusetts Institute of Technology, 6Department of Biological Engineering, Massachusetts Institute of Technology, 7Broad Institute Here, a simple, efficient, and cost-effective method of sgRNA cloning is outlined. Biology Subtype-specific Optical Action Potential Recordings in Human Induced Pluripotent Stem Cell-derived Ventricular Cardiomyocytes Alexander Goedel*1,2, Dorota M. Zawada*1, Fangfang Zhang1, Zhifen Chen3, Alessandra Moretti1,2, Daniel Sinnecker1,2 1Medical Department I, University Hospital Klinikum rechts der Isar, Technical University of Munich, 2German Centre for Cardiovascular Research (DZHK), Munich Heart Alliance, 3Beth Israel Deaconess Medical Center, Harvard Medical School Here we present a method to optically image action potentials, specifically in ventricular-like induced pluripotent stem cell-derived cardiomyocytes. The method is based on the promoter-driven expression of a voltage-sensitive fluorescent protein. Genetics Purification of Low-abundant Cells in the Drosophila Visual System Jing Peng1, Ivan J. Santiago1, Matthew Y. Pecot1 1Department of Neurobiology, Harvard Medical School Here, we present a cell dissociation protocol for efficiently isolating cells present at low abundance within the Drosophila visual system through fluorescence activated cell sorting (FACS). Medicine Home-Based Transcranial Direct Current Stimulation Device Development: An Updated Protocol Used at Home in Healthy Subjects and Fibromyalgia Patients Fabiana Carvalho1,2, Aline Patrícia Brietzke1,2, Assunta Gasparin1,2, Franciele Pereira dos Santos2,3, Rafael Vercelino4, Rafael Firmino Ballester2,3, Paulo Roberto Stefani Sanches5, Danton Pereira da Silva Jr5, Iraci L. S. Torres1,6, Felipe Fregni7,8, Wolnei Caumo1,2,9,10 1Post-Graduate Program in Medical Sciences, School of Medicine, Universidade Federal do Rio Grande do Sul (UFRGS), 2Laboratory of Pain & Neuromodulation, Hospital de Clínicas de Porto Alegre (HCPA), 3School of Medicine, Universidade Federal do Rio Grande do Sul (UFRGS), 4Faculdade de Desenvolvimento do Rio Grande do Sul (FADERGS), Health and Wellness School Laureate International Universities, 5Biomedical Engineering Department, Hospital de Clínicas de Porto Alegre (HCPA), 6Pharmacology Department, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul (UFRGS), 7Department of Physical Medicine and Rehabilitation, Harvard Medical School, 8Spaulding Neuromodulation Center, Spaulding Rehabilitation Hospital, 9Pain and Palliative Care Service, Hospital de Clínicas de Porto Alegre (HCPA), 10Department of Surgery, School of Medicine, Universidade Federal do Rio Grande do Sul (UFRGS) This study provides an updated home-based tDCS protocol that enables subjects to receive the beneficial effects of tDCS at home with an easy to use device with settings to control the use and dosage, enhancing the feasibility for long-term use at home. Biology Diagonal Method to Measure Synergy Among Any Number of Drugs Melike Cokol-Cakmak1, Feray Bakan2, Selim Cetiner1, Murat Cokol1,2,3 1Faculty of Engineering and Natural Sciences, Sabanci University, 2Nanotechnology Research and Application Center, Sabanci University, 3Laboratory of Systems Pharmacology, Harvard Medical School In this protocol, we describe how to make Loewe additivity-based drug interaction measurements for pairwise and three-way drug combinations. Immunology and Infection Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification Kourtney P. Nickerson1,2, Christina S. Faherty1,2 1Mucosal Immunology and Biology Research Center, Division of Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital, 2Department of Pediatrics, Harvard Medical School This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion. Immunology and Infection Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants Eliana Drenkard*1, Rhianna M. Hibbler*1, D. Alina Gutu2, Alexander D. Eaton1, Amy L. Silverio1, Frederick M. Ausubel2,3, Bryan P. Hurley1,4, Lael M. Yonker1,4 1Department of Pediatrics, Mucosal Immunology and Biology Research Center, Massachusetts General Hospital, 2Department of Molecular Biology, Massachusetts General Hospital, 3Department of Genetics, Harvard Medical School, 4Department of Pediatrics, Harvard Medical School Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library. Cancer Research Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells Nouran S. Abdelfattah1, Ann Mullally1,2,3 1 Targeted gene editing using CRISPR/Cas9 has greatly facilitated the understanding of the biological functions of genes. Here, we utilize the CRISPR/Cas9 methodology to model calreticulin mutations in cytokine-dependent hematopoietic cells in order to study their oncogenic activity. Biology Pulling Membrane Nanotubes from Giant Unilamellar Vesicles Coline Prévost*1,2,3, Feng-Ching Tsai*1,4, Patricia Bassereau1,4, Mijo Simunovic1,5 1Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University, CNRS UMR168, 2Department of Genetics and Complex Diseases, T. H. Chan School of Public Health, Harvard Medical School, 3Department of Cell Biology, Harvard Medical School, 4Sorbonne Universités, UPMC University Paris 06, 5Center for Studies in Physics and Biology, The Rockefeller University Many proteins in the cell sense and induce membrane curvature. We describe a method to pull membrane nanotubes from lipid vesicles to study the interaction of proteins or any curvature-active molecule with curved membranes in vitro. Behavior Stereotactically-guided Ablation of the Rat Auditory Cortex, and Localization of the Lesion in the Brain Verónica Lamas1,2, Sheila Estévez1, Marianni Pernía1, Ignacio Plaza1, Miguel A. Merchán1 1Institute of Neuroscience of Castilla y León, University of Salamanca, 2Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Department of Otolaryngology, Harvard Medical School We describe a method for the stereotactically-guided location, exposure, and ablation of the auditory cortex in rats. The localization of the ablation is assessed using a coordinate map postmortem. Developmental Biology The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging Nan Zhang1,2, Edward Membreno1, Susan Raj1, Hongjie Zhang1,3, Liakot A Khan1, Verena Gobel1 1Mucosal Immunology and Biology Research Center, Developmental Biology and Genetics Core, Massachusetts General Hospital for Children, Harvard Medical School, 2College of Life Sciences, Jilin University, 3Faculty of Health Sciences, University of Macau The C. elegans excretory canal is a unique single-cell model for the visual in vivo analysis of de novo polarized membrane biogenesis. This protocol describes a combination of standard genetic/RNAi and imaging approaches, adaptable for the identification and characterization of molecules directing unicellular tubulogenesis, and apical membrane and lumen biogenesis. Developmental Biology The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging Nan Zhang1,2, Liakot A Khan1, Edward Membreno1, Gholamali Jafari1, Siyang Yan1, Hongjie Zhang1,3, Verena Gobel1 1Mucosal Immunology and Biology Research Center, Developmental Biology and Genetics Core, Massachusetts General Hospital, Harvard Medical School, 2College of Life Sciences, Jilin University, 3Faculty of Health Sciences, University of Macau The transparent C. elegans intestine can serve as an "in vivo tissue chamber" for studying apicobasal membrane and lumen biogenesis at the single-cell and subcellular level during multicellular tubulogenesis. This protocol describes how to combine standard labeling, loss-of-function genetic/RNAi and microscopic approaches to dissect these processes on a molecular level. Bioengineering Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids Yu Shrike Zhang*1, Qingmeng Pi*1,2, Anne Metje van Genderen1,3 1Division of Engineering in Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 2Department of Plastic and Reconstructive Surgery, Renji Hospital, Shanghai Jiao Tong University School of Medicine, 3Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids. Cancer Research A Unified Methodological Framework for Vestibular Schwannoma Research Lukas D. Landegger*1,2,3, Jessica E. Sagers*1,4, Sonam Dilwali1,4, Takeshi Fujita1,2, Mehmet I. Sahin1,2, Konstantina M. Stankovic1,2,4 1Eaton Peabody Laboratories, Department of Otolaryngology, Massachusetts Eye and Ear, 2Department of Otolaryngology, Harvard Medical School, 3Department of Otolaryngology, Vienna General Hospital, Medical University of Vienna, 4Program in Speech and Hearing Bioscience and Technology, Harvard Medical School The goal of this protocol is to outline the collection and processing of human surgical samples for multiple downstream applications in vestibular schwannoma and Schwann cell research. Medicine Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research Lukas D. Landegger1,2,3, Sonam Dilwali1,4, Konstantina M. Stankovic1,2,4 1Eaton Peabody Laboratories, Department of Otolaryngology, Massachusetts Eye and Ear, 2Department of Otolaryngology, Harvard Medical School, 3Department of Otolaryngology, Vienna General Hospital, Medical University of Vienna, 4Harvard Program in Speech and Hearing Bioscience and Technology The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research. Biology Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules Anaïs Aulas*1,2, Marta M. Fay*1,2, Witold Szaflarski1,2,3, Nancy Kedersha1,2, Paul Anderson1,2, Pavel Ivanov1,2,4 1 Stress Granules (SGs) are nonmembranous cytoplasmic structures that form in cells exposed to a variety of stresses. SGs contain mRNAs, RNA-binding proteins, small ribosomal subunits, translation-related factors, and various cell signaling proteins. This protocol describes a workflow that uses several experimental approaches to detect, characterize, and quantify bona fide SGs. Developmental Biology A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening Charles A. C. Williams1, Nathanael S. Gray2,3, Greg M. Findlay1 1The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, 2Department of Cancer Biology, Dana-Farber Cancer Institute, 3Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School Here, we present a quantitative and scalable protocol to perform targeted small molecule screens for kinase regulators of the naïve-primed pluripotent transition. Medicine Two Techniques to Create Hypoparathyroid Mice: Parathyroidectomy Using GFP Glands and Diphtheria-Toxin-Mediated Parathyroid Ablation Ruiye Bi1,2, Yi Fan2,3, En Luo2,4, Quan Yuan2,4, Michael Mannstadt1 1Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, 2West China School of Stomatology, Sichuan University, 3Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, 4State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University Mice with acquired hypoparathyroidism would be useful for studying novel drug therapies for hypoparathyroidism. Two procedures to create such mice are demonstrated. The GFP-PTX mouse is generated by surgical parathyroidectomy guided by green fluorescing parathyroid glands. A second, non-surgical approach is based on parathyroid-specific expression of the diphtheria toxin receptor. Genetics Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins Alfredo J. Hernandez1, Charles C. Richardson1 1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School We describe sensitive, gel-based discontinuous assays to examine the kinetics of lagging-strand initiation using the replication proteins of bacteriophage T7. Genetics Detection of Copy Number Alterations Using Single Cell Sequencing Kristin A. Knouse1,2,3, Jie Wu4, Austin Hendricks5 1Koch Institute for Integrative Cancer Research, Department of Biology, Massachusetts Institute of Technology, 2Howard Hughes Medical Institute, 3Division of Health Sciences and Technology, Harvard Medical School, 4The Barbara K. Ostrom (1978) Bioinformatics and Computing Facility in the Swanson Biotechnology Center, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 5BioMicro Center, Department of Biology, Massachusetts Institute of Technology Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells. Neuroscience Measuring and Altering Mating Drive in Male Drosophila melanogaster Christine L. Boutros*1, Lauren E. Miner*1, Ofer Mazor2,3, Stephen X. Zhang3 1 This article describes a behavioral assay that uses male mating drive in Drosophila melanogaster to study motivation. Using this method, researchers can utilize advanced fly neurogenetic techniques to uncover the genetic, molecular, and cellular mechanisms that underlie this motivation. Genetics Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts Jian Ding1,2, Zhi-Qiang Lin1,2, Jian-Ming Jiang3,4, Christine E. Seidman3,4, Jonathan G. Seidman3,4, William T. Pu1,2, Da-Zhi Wang1,2 1 In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described. Developmental Biology Rapid Acquisition of 3D Images Using High-resolution Episcopic Microscopy Haochuan Zhang*1,2,3, JunGang Huang*2,3,4, Xin Liu2,3, Ping Zhu4, Zhongrong Li1, Xue Li2,3 1Department of Pediatric Surgery, The Second Affiliated Hospital & Yuying Children's Hospital, Wenzhou Medical University, 2 We describe a detailed protocol using high-resolution episcopic microscopy to acquire three-dimensional (3D) images of mouse embryos. This improved protocol utilizes a modified tissue preparation method to enhance penetration of the fluorescent dye, thereby permitting morphometric analysis of both small and large-sized specimens. Medicine A Multimodal Imaging- and Stimulation-based Method of Evaluating Connectivity-related Brain Excitability in Patients with Epilepsy Mouhsin M. Shafi1,2,3, Susan Whitfield-Gabrieli4, Catherine J. Chu1,5, Alvaro Pascual-Leone1,2,3, Bernard S. Chang1,2 1Department of Neurology, Harvard Medical School, 2Department of Neurology, Beth Israel Deaconess Medical Center, 3Berenson-Allen Center for Noninvasive Brain Stimulation, Beth Israel Deaconess Medical Center, 4Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, 5Department of Neurology, Massachusetts General Hospital Resting-state functional-connectivity MRI has identified abnormalities in patients with a wide range of neuropsychiatric disorders, including epilepsy due to malformations of cortical development. Transcranial Magnetic Stimulation in combination with EEG can demonstrate that patients with epilepsy have cortical hyperexcitability in regions with abnormal connectivity. Developmental Biology Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions Melinda L. Koyle1, Madeline Veloz1, Alec M. Judd1, Adam C.-N. Wong2,4, Peter D. Newell2,5, Angela E. Douglas2,3, John M. Chaston1,2 1Department of Plant and Wildlife Sciences, Brigham Young University, 2Department of Entomology, Cornell University, 3Department of Molecular Biology and Genetics, Cornell University, 4Division of Infectious Diseases, Boston Children's Hospital, Harvard Medical School, 5Biological Sciences, SUNY Oswego A method for rearing Drosophila melanogaster under axenic and gnotobiotic conditions is presented. Fly embryos are dechorionated in sodium hypochlorite, transferred aseptically to sterile diet, and reared in closed containers. Inoculating diet and embryos with bacteria leads to gnotobiotic associations, and bacterial presence is confirmed by plating whole-body Drosophila homogenates. Developmental Biology Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae Elliott J. Hagedorn1,2, Jennifer L. Cillis3, Caitlyn R. Curley3, Taylor C. Patch3, Brian Li1,2, Bradley W. Blaser1,2,7, Raquel Riquelme1,2, Leonard I. Zon1,2,4,5,6, Dhvanit I. Shah1,2,3,4,5 1Division of Hematology, Department of Medicine, Brigham and Women’s Hospital, 4Harvard Stem Cell Institute, 5Broad Institute of Massachusetts Institute of Technology, 6Howard Hughes Medical Institute, 7Division of Hematologic Malignancies, Dana-Farber Cancer Institute This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest. Medicine Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation Caitlin O'Rourke*1, Georgia Shelton*1,2, Joshua D. Hutcheson3,4, Megan F. Burke2, Trejeeve Martyn1, Timothy E. Thayer2, Hannah R. Shakartzi1, Mary D. Buswell1, Robert E. Tainsh1, Binglan Yu1,4, Aranya Bagchi1,4, David K. Rhee2,4, Connie Wu1,2,4, Matthias Derwall5, Emmanuel S. Buys1,4, Paul B. Yu3,4, Kenneth D. Bloch1,2,4, Elena Aikawa3,4, Donald B. Bloch1,5,6, Rajeev Malhotra2,4 1Anesthesia Center for Critical Care Research of the Department of Anesthesia, Critical Care, and Pain Medicine, Massachusetts General Hospital, 2Cardiovascular Research Center and Cardiology Division of the Department of Medicine, Massachusetts General Hospital, 3 Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging. Immunology and Infection Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions Achille Broggi1,2, Clara Cigni1, Ivan Zanoni1,2,3, Francesca Granucci1,3 1Department of Biotechnology and Biosciences, University of Milano-Bicocca, 2Boston Children's Hospital, Division of Gastroenterology, Harvard Medical School, 3Humanitas Clinical and Research Center The skin is home to a complex immune cell network. We describe an efficient methodology for the digestion of mouse skin, from different parts of the animal's body, in order to obtain a single-cell suspension and analyze the different leukocyte populations resident in the skin by flow cytometry. Immunology and Infection Induction of Maternal Immune Activation in Mice at Mid-gestation Stage with Viral Mimic Poly(I:C) Ke-Huan Chow*1, Zihao Yan*1,2, Wei-Li Wu1 1Division of Biology and Biological Engineering, California Institute of Technology, 2Harvard Medical School Maternal immune activation (MIA) is a model for an environmental risk factor of autism and schizophrenia. The goal of this article is to provide a step-by-step procedure of how to induce MIA in the pregnant mice in order to enhance the reproducibility of this model. Neuroscience A New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster Severine Trannoy1, Budhaditya Chowdhury1, Edward A. Kravitz1 1Department of Neurobiology, Harvard Medical School During fruit fly fights, the behavioral patterns observed, fight dynamics, and associated learning and memory are influenced by experimental conditions. The protocol presented here describes a novel procedure that entirely eliminates handling of flies during experiments. This improves fight dynamics and allows formation of strong "loser" effects. Medicine Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats Lauren L. Jantzie1,2, Jesse L. Winer3, Jessie R. Maxwell1, Lindsay A.S. Chan3, Shenandoah Robinson3,4 1Department of Pediatrics, University of New Mexico, 2Department of Neurosciences, University of New Mexico, 3 Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development. Developmental Biology Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel’s Cartilage Lucie J. Rochard*1, Irving T.C. Ling*1, Yawei Kong1, Eric C. Liao1 1Massachusetts General Hospital, Center for Regenerative Medicine, Harvard Medical School Cell organization of craniofacial bones has long been hypothesized but never directly visualized. Multi-spectral cell labeling and in vivo live imaging allows visualization of dynamic cell behavior in zebrafish lower jaw. Here, we detail the protocol to manipulate Zebrabow transgenic fish and directly observe cell intercalation and morphological changes of chondrocytes in the Meckel’s cartilage. Developmental Biology Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury Caramai N. Kamei1, Yan Liu1,2, Iain A. Drummond1,3 1Nephrology Division, Department of Medicine, Massachusetts General Hospital, 2Basic Sciences Division, Fred Hutchinson Cancer Research Center, 3Department of Genetics, Harvard Medical School Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons. Chemistry Self-assembly of Complex Two-dimensional Shapes from Single-stranded DNA Tiles Bryan Wei1, Michelle K. Vhudzijena2, Joanna Robaszewski2, Peng Yin2,3 1Tsinghua-Peking Center for Life Sciences, School of Life Sciences, Tsinghua University, 2Wyss Institute for Biologically Inspired Engineering, Harvard University, 3Department of Systems Biology, Harvard Medical School DNA tiling is an effective approach to make programmable nanostructures. We describe the protocols to construct complex two-dimensional shapes by the self-assembly of single-stranded DNA tiles. Behavior Protocol for Studying Extinction of Conditioned Fear in Naturally Cycling Female Rats Lisa Y. Maeng1,2, Kara K. Cover1, Aaron J. Landau1, Mohammed R. Milad1,2, Kelimer Lebron-Milad1,2 1Department of Psychiatry, Massachusetts General Hospital, 2Harvard Medical School Gonadal hormones such as estrogen modulate memory formation in a number of experimental paradigms including fear extinction memory. This protocol describes a set of methods for investigating the influence of gonadal hormones specifically during extinction in naturally cycling females, including estrous cycle monitoring and exogenous estrogen administration. Medicine Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres Shannon Bunker1, Joanna Holeniewska1, Sauparnika Vijay2, Annegret Dahlmann-Noor2,3, Peng Khaw2,4, Yin-Shan Ng5, David Shima1,6, Richard Foxton1 1Ocular Biology and Therapeutics, University College London Institute of Ophthalmology, 2University College London Institue of Ophthalmology, 3Moorfields Eye Hospital, 4NIHR Biomedical Research Centre, Moorfields Eye Hospital, 5Schepens Eye Research Institute, Harvard Medical School, 6Hoffman-La Roche We present a method for inducing elevated intraocular pressure (IOP), by injecting magnetic microspheres into the rat eye, to model glaucoma. This leads to strong pressure rises, and extensive neuronal death. This protocol is easy to perform, does not require repeat injections, and produces stable long-lasting IOP rises. Neuroscience Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway Elliott D. Kozin*1, Keith N. Darrow*2, Ariel E. Hight*1, Ashton E. Lehmann1, Alyson B. Kaplan1, M. Christian Brown1, Daniel J. Lee1 1Department of Otology and Laryngology, Harvard Medical School, 2Department of Communication Sciences and Disorders, Worcester State University The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model. Biology Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9 Daniel E. Bauer*1,2,3, Matthew C. Canver*1, Stuart H. Orkin1,2,3,4 1Harvard Medical School, 2 CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9. Bioengineering Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay Lama Mulki*1,2, J. Harry Sweigard*1, Kip M. Connor1 1Angiogenesis Laboratory, Department of Ophthalmology, Harvard Medical School, Massachusetts Eye & Ear Infirmary, 2 Here, we present a protocol that allows the investigator to assess leukocyte recruitment dynamics ex vivo by connecting a chamber coated with endothelial-derived adhesion molecules to the circulatory system of a mouse. This method offers significant advantages since it allows for leukocyte assessment under relative biological conditions. Biology Assessing Cerebral Autoregulation via Oscillatory Lower Body Negative Pressure and Projection Pursuit Regression J. Andrew Taylor1,2, Can Ozan Tan1,2, J. W. Hamner2 1Department of Physical Medicine and Rehabilitation, Harvard Medical School, 2Cardiovascular Research Laboratory, Spaulding Hospital Cambridge Cerebral perfusion is maintained across a range of pressures via cerebral autoregulation. However, characterizing autoregulation requires prominent pressure fluctuations at regulated frequencies. The described protocol will show how oscillatory lower body negative pressure can generate pressure fluctuations to provide data for projection pursuit regression for quantification of the autoregulatory curve. Biology Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology William S. Phipps*1, Zhizhong Yin*1, Candice Bae1, Julia Z. Sharpe1, Andrew M. Bishara2, Emily S. Nelson3, Aaron S. Weaver3, Daniel Brown4, Terri L. McKay3, DeVon Griffin3, Eugene Y. Chan1 1DNA Medicine Institute, 2Harvard Medical School, 3NASA Glenn Research Center, 4ZIN Technologies Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups. Neuroscience The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye Amy E. Birsner*1, Ofra Benny*2, Robert J. D'Amato1,3 1 The protocol describes the corneal micropocket assay as developed in mice. Medicine Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice Richie E. Kohman1, Xue Han1, Benjamin S. Bleier2 1Department of Biomedical Engineering, Boston University, 2Department of Otology and Laryngology, Massachusetts Eye and Ear Infirmary, Harvard Medical School A mouse model of human endoscopic skull base reconstruction has been developed that creates a semipermeable interface between the brain and nose using nasal mucosal grafts. This method allows researchers to study delivery to the central nervous system of high molecular weight therapeutics which are otherwise excluded by the blood-brain barrier when administered systemically. Bioengineering Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood Caroline N. Jones*1,2,3, Anh N. Hoang*1,2,3, Laurie Dimisko1, Bashar Hamza1, Joseph Martel1,4, Daniel Irimia1,2,3 1The BioMEMS Resource Center, Department of Surgery, Massachusetts General Hospital, 2Harvard Medical School, 3Shriners Burns Hospital, 4Harvard University School of Engineering and Applied Sciences This protocol details an assay designed to measure human neutrophil chemotaxis from one droplet of whole blood with robust reproducibility. This approach circumvents the need for neutrophil separation and requires only a few minutes of assay preparation time. The microfluidic chip enables the repeated measure of neutrophil chemotaxis over time in infants or small mammals, where sample volume is limited. Bioengineering Manufacture of Concentrated, Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration Lindsay M. Thomson1, Brian D. Polizzotti1, Frances X. McGowan2, John N. Kheir1 1Department of Cardiology, Boston Children's Hospital, Harvard Medical School, 2Department of Anesthesiology and Critical Care, Children's Hospital of Philadelphia, University of Pennsylvania We describe methods for the manufacture of large volumes of lipid-based oxygen microbubbles (LOMs) designed for intravenous oxygen delivery using high-shear homogenization and serial concentration. Medicine Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair Dongsu Park1, Joel A. Spencer2, Charles P. Lin2, David T. Scadden1 1Center for Regenerative Medicine, Massachusetts General Hospital, Harvard Stem Cell Institute, 2Wellman Center for Photomedicine and Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture. Environment Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun Matthew D. Mattozzi1,2, Mathias J. Voges1,2,3, Pamela A. Silver1,2, Jeffrey C. Way1,2 1Synthetic Biology Platform, Wyss Institute for Biologically Inspired Engineering, Harvard University, 2Department of Systems Biology, Harvard Medical School, 3Department of Biotechnology, Delft University of Technology This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun. Biology Nanopodia - Thin, Fragile Membrane Projections with Roles in Cell Movement and Intercellular Interactions Chi-Iou Lin1, Chun-Yee Lau1, Dan Li1, Shou-Ching Jaminet1 1Center for Vascular Biology Research, Department of of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School Nanopodia are thin but fragile membrane channels that extend up to 100 μm from a cell's leading front or trailing rear and sense the cellular environment. Direct fixation at 37 °C, gentle washing, and avoidance of organic solvents like ethanol, methanol, or acetone and of higher Triton X-100 concentrations are required to observe these cellular structures. Neuroscience Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans Limin Hao1,2, Edgar A. Buttner1,2 1Department of Psychiatry, Harvard Medical School, 2Mailman Research Center, McLean Hospital Approaches for testing the effects of antipsychotic drugs (APDs) in Caenorhabditis elegans are demonstrated. Assays are described for testing drug effects on development and viability and on pharyngeal pumping rate. These methods are also applicable for pharmacogenetic experiments with drug classes other than APDs. Medicine Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography Hui-Wen Cheng*1,2, Sudeshna Fisch*1, Susan Cheng1, Michael Bauer1, Soeun Ngoy1, Yiling Qiu1, Jian Guan1, Shikha Mishra1, Christopher Mbah1, Ronglih Liao1 1Cardiac Muscle Research Labratory, Cardiovascular Division, Brigham and Women’s Hospital, Harvard Medical School, 2Cardiovascular Department, Chang Gung Memorial Hospital Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload. Medicine Dried Blood Spot Collection of Health Biomarkers to Maximize Participation in Population Studies Michael W. Ostler1, James H. Porter1,2, Orfeu M. Buxton1,2,3,4 1Center for Population and Development Studies, Harvard School of Public Health, 2 Biomarkers are directly-measured biological indicators of disease or health. In population and social sciences, biomarkers need to be easy to obtain, transport, and analyze. Dried Blood Spot (DBS) collection meets this need, can be collected in the field with high response rates and analyzed for a variety of biomarkers. Neuroscience Isolation and Culture of Endothelial Cells from the Embryonic Forebrain Peeyush Kumar T.1,2, Anju Vasudevan1,2 1Department of Psychiatry, Harvard Medical School, 2Angiogenesis and Brain Development Laboratory, Division of Basic Neuroscience, McLean Hospital This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis. Bioengineering An Improved Method for the Preparation of Type I Collagen From Skin Christina A. Pacak1,2, Allison A. MacKay1, Douglas B. Cowan1,2 1 Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr. Medicine Ultrasonic Assessment of Myocardial Microstructure Pranoti Hiremath1, Michael Bauer2, Hui-Wen Cheng2, Kazumasa Unno2, Ronglih Liao2, Susan Cheng2 1Harvard Medical School, 2 Echocardiography is commonly used to noninvasively characterize and quantify changes in cardiac structure and function. We describe an ultrasound-based imaging algorithm that offers an enhanced surrogate measure of myocardial microstructure and can be performed using open-access image analysis software. Bioengineering Stress-induced Antibiotic Susceptibility Testing on a Chip Maxim Kalashnikov1, Jennifer Campbell1, Jean C. Lee2, Andre Sharon1,3, Alexis F. Sauer-Budge1,4 1Fraunhofer USA Center for Manufacturing Innovation, 2Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 3Department of Mechanical Engineering, Boston University, 4Department of Biomedical Engineering, Boston University We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions. Immunology and Infection In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration Mark E. Kusek1,2,3, Michael A. Pazos1,3, Waheed Pirzai3, Bryan P. Hurley1,3 1Department of Pediatrics, Harvard Medical School, 2Department of Pediatric Gastroenterology, MGH for Children, 3Mucosal Immunology and Biology Research Center, Massachusetts General Hospital Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease. An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon. Biology Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells Susan Fotheringham1,2, Keren Levanon1,2,3, Ronny Drapkin1,2,4 1Department of Medical Oncology, Dana-Farber Cancer Institute, 2Harvard Medical School, Boston, MA, 3Sheba Cancer Research Center, Chaim Sheba Medical Center, 4Department of Pathology, Brigham and Women's Hospital The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.
