Brigham and Women's Hospital, Harvard Medical School 24 articles published in JoVE Biology Polycarbonate Ultracentrifuge Tube Re-Use in Proteomic Analyses of Extracellular Vesicles Rachel M. E. Cahalane*1,2, Mandy E. Turner*1, Cassandra L. Clift1, Mark C. Blaser1, Gabrielle Bogut1, Sydney Levy1, Taku Kasai1, Tom A. P. Driedonks3,4, Esther N. M. Nolte-‘t Hoen4, Masanori Aikawa1,5, Sasha A. Singh1,5, Elena Aikawa1,5 1Center for Excellence in Vascular Biology, Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School A detailed protocol is provided for cleaning and re-using polycarbonate ultracentrifuge tubes to perform extracellular vesicle isolation suitable for proteomics experiments. Bioengineering CRISPR-Cas-mediated Multianalyte Synthetic Urine Biomarker Test for Portable Diagnostics Audrey E. Van Heest1, Feiyang Deng1, Renee T. Zhao2, Nour Saida Harzallah2,3, Heather E. Fleming3,4, Sangeeta N. Bhatia2,3,4,5,6,7,8, Liangliang Hao1,2,3 1Department of Biomedical Engineering, Boston University, 2Institute for Medical Engineering and Science, Massachusetts Institute of Technology, 3Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 4Howard Hughes Medical Institute, 5Broad Institute of Massachusetts Institute of Technology and Harvard, 6Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, 7Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, 8 This protocol describes a CRISPR-Cas-mediated, multianalyte synthetic urine biomarker test that enables point-of-care cancer diagnostics through the ex vivo analysis of tumor-associated protease activities. Neuroscience Three-dimensional Navigation-guided, Prone, Single-position, Lateral Lumbar Interbody Fusion Technique Samantha E. Hoffman1, Saksham Gupta1, Kevin Huang1, Neil Klinger1, Yi Lu1 1 The single-position, prone, lateral approach allows for both lateral lumbar interbody placement and direct posterior decompression with pedicle screw placement in one position. Developmental Biology Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity Yvon Woappi1,2, Geraldine Ezeka3, Justin Vercellino4, Sean M. Bloos5, Kim E. Creek6, Lucia Pirisi1 1Department of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, 2 Here we describe a protocol for the systematic cultivation of epidermal spheroids in 3D suspension culture. This protocol has wide-ranging applications for use in a variety of epithelial tissue types and for the modeling of several human diseases and conditions. Immunology and Infection Fecal (micro) RNA Isolation Fyonn H. Dhang1,2, Howard L. Weiner1,2, Shirong Liu1,2 1 This protocol isolates high quality total RNA from fecal samples of animal and human subjects. A commercial miRNA isolation kit is used with significant adaption to isolate pure RNA with optimized quantity and quality. The RNA isolates are good for most downstream RNA assays such as sequencing, micro-array, and RT-PCR. Developmental Biology High-Frequency Ultrasound Echocardiography to Assess Zebrafish Cardiac Function Alessandro Evangelisti1, Katharina Schimmel1, Shaurya Joshi2, Kavya Shah1, Sudeshna Fisch2, Kevin M. Alexander1, Ronglih Liao1, Isabel Morgado1 1Stanford Cardiovascular Institute, Stanford University, 2 We describe a protocol to assess heart morphology and function in adult zebrafish using high-frequency echocardiography. The method allows visualization of the heart and subsequent quantification of functional parameters, such as heart rate (HR), cardiac output (CO), fractional area change (FAC), ejection fraction (EF), and blood inflow and outflow velocities. Biology Quantification of Proliferative and Dead Cells in Enteroids Hua-Shan Li*1, Shao-Fang Xu*1, Jian-Ying Sheng*1, Zhi-Hui Jiang1, Jing Wang1, Ning Ding1, Tao Wang1, Matthew A. Odenwald2, Jerrold R. Turner2,3, Wei-Qi He1, Hong Xu1, Juan-Min Zha1 1Jiangsu Key Laboratory of Neuropsychiatric Diseases and Cambridge-Suda (CAM-SU) Genomic Resource Center, Medical College of Soochow University, Department of Oncology, The First Affiliated Hospital of Soochow University, 2Department of Pathology, University of Chicago, 3 The presented protocol uses flow cytometry to quantify the number of proliferating and dead cells in cultured mouse enteroids. This method is helpful to evaluate the effects of drug treatment on organoid proliferation and survival. Genetics Characterization of Functionally Associated miRNAs in Glioblastoma and their Engineering into Artificial Clusters for Gene Therapy Vivek Bhaskaran1, Pierpaolo Peruzzi1 1 Described here is a protocol for characterizing modules of biologically synergistic miRNAs and their assembly into short transgenes, which allows simultaneous overexpression for gene therapy applications. Immunology and Infection A Model of Self-limited Acute Lung Injury by Unilateral Intra-bronchial Acid Instillation Alexander H. Tavares1, Jennifer K. Colby1, Bruce D. Levy1, Raja-Elie Edward Abdulnour1 1 Selective intra-bronchial acid instillation to the left lung in mice results in unilateral and self-limited acute lung injury that models human acute respiratory distress syndrome (ARDS) induced by gastric acid aspiration. Biology Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy Rafael M. Rezende*1, Mateus E. Lopes*2, Gustavo B Menezes2, Howard L. Weiner1 1 This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure. Medicine Surgical Training for the Implantation of Neocortical Microelectrode Arrays Using a Formaldehyde-fixed Human Cadaver Model Pierre Mégevand1,2, Alain Woodtli1, Aude Yulzari1, G. Rees Cosgrove3, Shahan Momjian4, Bojan V. Stimec5, Marco V. Corniola4, Jean H. D. Fasel5 1Wyss Center for Bio and Neuroengineering, Geneva, 2Division of Neurology, Department of Clinical Neuroscience, Geneva University Hospitals, 3 We designed a procedure in which a formaldehyde-fixed human cadaver is used to assist neurosurgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain. Medicine Early Detection of Drug-Induced Renal Hemodynamic Dysfunction Using Sonographic Technology in Rats Sudeshna Fisch1, Ronglih Liao1, Li-Li Hsiao2, Tzongshi Lu2 1Renal Division, Brigham and Women's Hospital, Harvard Medical School Early stage hemodynamic dysfunction is critical to the development of kidney disease. Yet, detection methodologies are limited. Recent advances in sonography provide a noninvasive, accurate option for early detection of kidney injury. This study outlines a step-by-step, sonographic methodology for detecting kidney dysfunction using a drug-induced nephrotoxicity rat model. Medicine A Multicenter MRI Protocol for the Evaluation and Quantification of Deep Vein Thrombosis Venkatesh Mani1, Nadia Alie1, Sarayu Ramachandran1, Philip M. Robson1, Cecilia Besa1, Gregory Piazza2, Michele Mercuri3, Michael Grosso3, Bachir Taouli1, Samuel Z. Goldhaber2, Zahi A. Fayad1 1Department of Radiology, Translational and Molecular Imaging Institute, Icahn School of Medicine at Mount Sinai, 2 The goal of this study is to use magnetic resonance venography with long-circulating gadolinium-based contrast agent and direct thrombus imaging for quantitative evaluation of DVT volume in a multicenter, clinical trial setting. Inter- and intra-observer variability assessments were conducted, and reproducibility of the protocol was determined. Medicine Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice Wei-Ting Chang*1,2, Sudeshna Fisch*1, Michael Chen1, Yiling Qiu1, Susan Cheng1, Ronglih Liao1 1 Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding. Neuroscience Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus Tharakeswari Selvakumar1, Kambiz N. Alavian2, Travis Tierney1 1 The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery. Neuroscience Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains Maria Weinert1, Tharakeswari Selvakumar2, Travis S. Tierney2, Kambiz N. Alavian1,3 1Division of Brain Sciences, Department of Medicine, Imperial College London, 2 The causes of degeneration of midbrain dopaminergic neurons during Parkinson’s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration. Chemistry Preparation and Characterization of SDF-1α-Chitosan-Dextran Sulfate Nanoparticles Andrew R. Bader1, Tina Li1, Weiping Wang2, Daniel S. Kohane2, Joseph Loscalzo1, Ying-Yi Zhang1 1Laboratory for Biomaterials and Drug Delivery, Department of Anesthesiology, Division of Critical Care, Boston Children's Hospital The objective of this protocol is to incorporate SDF-1α, a stem cell homing factor, into dextran sulfate-chitosan nanoparticles. The resultant particles are measured for their size and zeta potential, as well as the content, activity, and in vitro release rate of SDF-1α from the nanoparticles. Medicine Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice Maria E. Laucho-Contreras1, Katherine L. Taylor1, Ravi Mahadeva2, Steve S. Boukedes3, Caroline A. Owen1,4 1 The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice. Immunology and Infection Detection of True IgE-expressing Mouse B Lineage Cells Michael P. Gallagher*1, Akritee Shrestha*1, Jennifer M. Magee1, Duane R. Wesemann1 1 In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining. Medicine Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement Alban Longchamp*1, Florent Allagnat*2, Xavier Berard3, Florian Alonso2, Jacques-Antoine Haefliger2, Sébastien Deglise*4, Jean-Marc Corpataux*4 1 The mechanisms leading to the development of intimal hyperplasia (IH) and vein graft failure are still poorly understood. This study describes an ex vivo system to perfuse human veins under controlled flow and pressure. Furthermore the efficiency of external mesh reinforcement to limit the development of IH was evaluated. Immunology and Infection Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice Ilias I. Siempos1,3, Hilaire C. Lam1, Yan Ding2, Mary E. Choi2, Augustine M. K. Choi1, Stefan W. Ryter1 1 Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method. Current protocols to assess autophagy in vivo in the context of CLP-induced sepsis are presented here: A protocol for measuring autophagy using (GFP)-LC3 mice, and a protocol for measuring autophagosome formation by electron microscopy. 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 Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow Monica M. Burdick1,2, Nathan M. Reynolds1,2, Eric W. Martin2, Jacquelyn V. Hawes1, Grady E. Carlson1, Chaz M. Cuckler1, Michael C. Bates1, Steven R. Barthel3, Charles J. Dimitroff3 1Department of Chemical and Biomolecular Engineering, Ohio University, 2Biomedical Engineering Program, Russ College of Engineering and Technology, Ohio University, 3 Compared with traditional affinity chromatography using protein A agarose bead-packed columns, protein A membrane adsorbers can significantly speed laboratory-scale isolation of antibodies and other Fc fragment-expressing proteins. Appropriate analysis and quantification methods can further accelerate protein processing, allowing isolation/characterization to be completed in one workday, instead of 20+ work hours. Biology Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation Maria L. Lombardi1, Monika Zwerger1, Jan Lammerding1,2 1 We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.
