Massachusetts General Hospital View Institution's Website 57 articles published in JoVE 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 Focused Assessment with Sonography for Trauma (FAST) Exam: Image Acquisition John D. Ritchie1, Charles N. Trujillo2, David L. Convissar3, William Shihao Lao4, Sean Montgomery5, Yuriy S. Bronshteyn6 1Trauma, Acute, and Critical Care Surgery, Duke University Hospital, 2Mount Sinai Medical Center, 3Department of Anesthesiology, Massachusetts General Hospital, 4Acute Care Surgery, Atrium Health, 5Department of Surgery, Duke University School of Medicine, 6Department of Anesthesiology, Duke University School of Medicine, Duke University Health System, Durham Veterans Health Administration The Focused Assessment with Sonography for Trauma (FAST) exam is a diagnostic point-of-care ultrasound examination used to screen for the presence of free fluid in the pericardium and peritoneum. Indications, techniques, and pitfalls of the procedure are discussed in this article. Medicine Gastric Point of Care Ultrasound in Adults: Image Acquisition and Interpretation Eric R. Heinz1, Omar Al-Qudsi2, David L. Convissar3, Marianne D. David1, Jennifer E. Dominguez2, Stephen Haskins4, Christina Jelly5, Anahi Perlas6, Anita N. Vincent1, Yuriy S. Bronshteyn2 1Department of Anesthesiology and Critical Care Medicine, George Washington University, 2Department of Anesthesiology, Duke University School of Medicine, Duke University Health System, 3Department of Anesthesiology, Massachusetts General Hospital, 4Department of Anesthesiology, Critical Care & Pain Management, Hospital for Special Surgery, 5Department of Anesthesiology, Vanderbilt University Medical Center, 6Department of Anesthesia and Pain Management, Toronto Western Hospital, University Health Network This protocol introduces two methods for image acquisition in gastric ultrasonography. Additionally, tips are provided for interpreting this information to assist in medical decision-making. 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. Medicine Point-of-Care Lung Ultrasound in Adults: Image Acquisition Rodrigo O. L. Pereira1, David L. Convissar2, Sean Montgomery3, James T. Herbert1, Christopher R. Reed3, Hoang J. Tang3, Yuriy S. Bronshteyn1,4 1Department of Anesthesiology, Duke University School of Medicine, 2Department of Anesthesiology, Massachusetts General Hospital, 3Department of Surgery, Duke University School of Medicine, Duke University Health System, 4Department of Anesthesiology, Durham VA Point-of-care ultrasound (POCUS) of the lungs provides quick answers in rapidly changing clinical scenarios. We present an efficient and informative protocol for image acquisition for use in acute care settings. 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. Medicine Point-Of-Care Ultrasound Screening for Proximal Lower Extremity Deep Venous Thrombosis Rebecca G. Theophanous1, Vinca W. Chow2, David L. Convissar3, Stephen C. Haskins4,5, Robert A. Jones6, Hari K. P. Kalagara7, Yuriy S. Bronshteyn8 1Department of Emergency Medicine, Duke University School of Medicine, 2Department of Anesthesiology, Dartmouth-Hitchcock Medical Center, 3Department of Anesthesiology, Massachusetts General Hospital, 4Department of Anesthesiology, Critical Care and Pain Management, Hospital for Special Surgery, 5Department of Anesthesiology, Weill-Cornell Medical College, 6Department of Emergency Medicine, MetroHealth Medical Center/Case Western Reserve University, 7Department of Anesthesiology, Mayo Clinic, 8Department of Anesthesiology, Duke University School of Medicine Traditionally, lower extremity deep venous thrombosis (DVT) is diagnosed by radiology-performed venous duplex ultrasound. Providers appropriately trained in focused point-of-care ultrasound (POCUS) can perform a rapid bedside examination with high sensitivity and specificity in critically ill patients. We describe the scanning technique for focused POCUS DVT lower extremity examination. 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. Medicine Image Acquisition Method for the Sonographic Assessment of the Inferior Vena Cava Maximilian Hoffman1, David L. Convissar2, Marie-Louise Meng1, Sean Montgomery4, Yuriy S. Bronshteyn1,3 1Department of Anesthesiology, Duke University School of Medicine, 2Department of Anesthesiology, Massachusetts General Hospital, 3Department of Anesthesiology, Durham VA, 4Department of Surgery, Duke University School of Medicine Point-of-care ultrasound evaluation of the inferior vena cava (IVC) is commonly utilized to identify, among other things, the volume status. Imaging should be performed systematically to ensure repeatability. This manuscript reviews the methods and pitfalls of sonographic IVC examination. 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. Medicine Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome Jacob Vieira1, Tara Gallagher2, Hui-Yu Sui1, Sirus Jesudasen3, Katrine Whiteson2, George A. O'Toole4, Kurt Hanselmann5, Peggy S. Lai1 1Division of Pulmonary and Critical Care Medicine, Massachusetts General Hospital, 2Department of Molecular Biology & Biochemistry, University of California, Irvine, 3Department of Medicine, Massachusetts General Hospital, 4Department of Microbiology & Immunology, Geisel School of Medicine at Dartmouth, 5Swiss i-research and teaching institute The goal of this protocol is to develop a model system for the effect of hyperoxia on cystic fibrosis airway microbial communities. Artificial sputum medium emulates the composition of sputum, and hyperoxic culture conditions model the effects of supplemental oxygen on lung microbial communities. 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. 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. 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 Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy Eriko Katsuta1, Masanori Oshi1, Omar M. Rashid2,3,4,5, Kazuaki Takabe1,6,7,8,9,10 1Breast Surgery, Department of Surgical Oncology, Roswell Park Comprehensive Cancer Center, 2Holy Cross Hospital Michael and Dianne Bienes Comprehensive Cancer Center, 3Department of Surgery, Massachusetts General Hospital, 4Department of Surgery, University of Miami Miller School of Medicine, 5Department of Surgery, Nova Southeastern University School of Medicine, 6Department of Surgery, University at Buffalo Jacobs School of Medicine and Biomedical Sciences, 7Department of Breast Surgery and Oncology, Tokyo Medical University, 8Department of Surgery, Yokohama City University, 9Department of Surgery, Niigata University Graduate School of Medical and Dental Sciences, 10Department of Surgery, Fukushima Medical University We introduce a murine orthotopic breast cancer model and radical mastectomy model with bioluminescence technology to quantify the tumor burden to mimic human breast cancer progression. 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. 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. Medicine In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice Caryn Porter1, Maria Ennamorati2, Nitya Jain2 1Center for Computational and Integrative Biology, Massachusetts General Hospital, 2Mucosal Immunology and Biology Research Center, Massachusetts General Hospital The protocol described here utilizes a photolabeling approach in newborn mice to specifically identify immune cells that emigrate from the colon to extra-intestinal sites. This strategy will be useful to study host-microbiome interactions in early life. 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. Bioengineering Preparation, Purification, and Use of Fatty Acid-containing Liposomes Lin Jin*1,2, Aaron E. Engelhart*1,3, Katarzyna P. Adamala1,3, Jack W. Szostak1 1Howard Hughes Medical Institute and Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, 2Department of Biomedical Engineering, Boston University, 3Department of Genetics, Cell Biology, and Development, University of Minnesota Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles. 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. 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. Biology Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization Melis N. Anahtar1, Brittany A. Bowman1, Douglas S. Kwon1 1Ragon Institute of MGH, MIT, and Harvard, Massachusetts General Hospital We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes. Medicine Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms Manav Korpal1, Jacob Feala1, Xiaoling Puyang1, Jian Zou1, Alex H. Ramos2, Jeremy Wu1, Timm Baumeister1, Lihua Yu1, Markus Warmuth1, Ping Zhu1 1H3 Biomedicine, 2Massachusetts General Hospital Drug resistance to targeted therapeutics is widespread and the need to identify mechanisms of resistance--prior to or following clinical onset--is critical for guiding alternative clinical management strategies. Here, we present a protocol to couple derivation of drug-resistant lines in vitro with sequencing to expedite discovery of these mechanisms. 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. 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. Immunology and Infection Normal and Malignant Muscle Cell Transplantation into Immune Compromised Adult Zebrafish Inês M. Tenente*1,2,3, Qin Tang*1,2, John C. Moore1,2, David M. Langenau1,2 1Molecular Pathology, Cancer Center and Center for Regenerative Medicine, Massachusetts General Hospital, 2Harvard Stem Cell Institute, 3GABBA - Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto Here, we present a protocol for cell transplantation of zebrafish skeletal muscle and embryonal rhabdomyosarcoma (ERMS) into adult immune compromised rag2E450fs homozygous mutant zebrafish. This protocol allows for the efficient analysis of regeneration and malignant transformation of muscle cells. Medicine Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue Xueli Zhang1,2, Chaincy Kuo3, Anna Moore1, Chongzhao Ran1 1Molecular Imaging Laboratory, MGH/MIT/HMS Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital/Harvard Medical School, 2Center for Drug Discovery, School of Pharmacy, China Pharmaceutical University, 3Perkin Elmer In this video report, we show the application of Cerenkov Luminescence Imaging (CLI) for interscapular brown adipose tissue in mice under activated and depressed conditions. Chemistry Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling Andrew C. Tolonen1,2,3, Wilhelm Haas4 1CEA, DSV, IG, Genoscope, 2CNRS-UMR8030, Évry, France, 3Université d'Évry Val d'Essonne, 4Massachusetts General Hospital Cancer Center Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a rapid, inexpensive strategy for accurate mass spectrometry-based quantitative proteomics. Here we demonstrate a robust method for preparation and analysis of protein mixtures using the ReDi approach that can be applied to nearly any sample type. 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. Biology High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay Jessica M. Baker1, Frederick M. Boyce1 1Department of Neurology, Massachusetts General Hospital We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest. 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 Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy Lisa Gfrerer1, Max Dougherty1, Eric C. Liao1 1Center for Regenerative Medicine, Massachusetts General Hospital Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish. Chemistry Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications Carlos Tassa1, Monty Liong1, Scott Hilderbrand1, Jason E. Sandler1, Thomas Reiner1, Edmund J. Keliher1, Ralph Weissleder1, Stanley Y. Shaw1 1Center for Systems Biology, Massachusetts General Hospital We present a method for rapid, reversible immobilization of small molecules and functionalized nanoparticle assemblies for Surface Plasmon Resonance (SPR) studies, using sequential on-chip bioorthogonal cycloaddition chemistry and antibody-antigen capture. Behavior Creating Dynamic Images of Short-lived Dopamine Fluctuations with lp-ntPET: Dopamine Movies of Cigarette Smoking Evan D. Morris1,2,3,4, Su Jin Kim1,3, Jenna M. Sullivan1,3,4, Shuo Wang3,4, Marc D. Normandin5, Cristian C. Constantinescu6, Kelly P. Cosgrove1,2,3 1Diagnostic Radiology, Yale University, 2Psychiatry, Yale University, 3Yale PET Center, Yale University, 4Biomedical Engineering, Yale University, 5Nuclear Medicine, Massachusetts General Hospital, 6Radiological Sciences, University of California, Irvine We present a novel PET imaging approach for capturing dopamine fluctuations induced by cigarette smoking. Subjects smoke in the PET scanner. Dynamic PET images are modeled voxel-by-voxel in time by lp-ntPET, which includes a time-varying dopamine term. The results are 'movies' of dopamine fluctuations in the striatum during smoking. Biology Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs Judith A. Sharp1,2, Mike D. Blower1,2 1Department of Molecular Biology, Massachusetts General Hospital, 2Department of Genetics, Harvard Medical School We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs. Immunology and Infection New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals Mathieu Angin1, Melanie King1, Marylyn Martina Addo1,2 1Ragon Institute of MGH, MIT, and Harvard, 2Division of Infectious Diseases, Massachusetts General Hospital CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals. Bioengineering Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation Lida P. Hariri1,2,3, Matthew B. Applegate4, Mari Mino-Kenudson1,2, Eugene J. Mark1,2, Brett E. Bouma2,3, Guillermo J. Tearney1,2,3, Melissa J. Suter2,3,5 1Department of Pathology, Harvard Medical School, 2Massachusetts General Hospital, 3Wellman Center for Photomedicine, Harvard Medical School, 4Pulmonary and Critical Care Unit, Massachusetts General Hospital, 5Pulmonary and Critical Care Unit, Harvard Medical School A method to image ex vivo pulmonary resection specimens with optical frequency domain imaging (OFDI) and obtain precise correlation to histology is described, which is essential to developing specific OFDI interpretation criteria for pulmonary pathology. This method is applicable to other tissue types and imaging techniques to obtain precise imaging to histology correlation for accurate image interpretation and assessment. Imaging criteria established with this technique would then be applicable to image assessment in future in vivo studies. Biology A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning Jamie Freeman1, Janos Kriston-Vizi1, Brian Seed2, Robin Ketteler1 1MRC LMCB, University College London, 2Center for Computational and Integrative Biology, Massachusetts General Hospital A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells. Medicine How to Measure Cortical Folding from MR Images: a Step-by-Step Tutorial to Compute Local Gyrification Index Marie Schaer1, Meritxell Bach Cuadra2,3, Nick Schmansky4, Bruce Fischl4, Jean-Philippe Thiran2, Stephan Eliez1 1Department of Psychiatry, University of Geneva School of Medicine, 2Signal Processing Laboratory, École Polytechnique Fédérale de Lausanne, 3Department of Radiology, University Hospital Center and University of Lausanne, 4Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital Measuring gyrification (cortical folding) at any age represents a window into early brain development. Hence, we previously developed an algorithm to measure local gyrification at thousands of points over the hemisphere1. In this paper, we detail the computation of this local gyrification index. Biology Transplantation of Cells Directly into the Kidney of Adult Zebrafish Cuong Q. Diep1, Alan J. Davidson1 1Center for Regenerative Medicine, Massachusetts General Hospital Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish. Medicine Intra-Operative Behavioral Tasks in Awake Humans Undergoing Deep Brain Stimulation Surgery John T. Gale*1,2, Clarissa Martinez-Rubio*1,2, Sameer A. Sheth1,2, Emad N. Eskandar1,2 1Nayef Al-Rodhan Laboratories for Cellular Neurosurgery and Neurosurgical Technology, Harvard Medical School, 2Department of Neurosurgery , Massachusetts General Hospital Deep brain stimulation surgery offers a unique opportunity to examine information encoding in the awake human brain. This article will describe intra-operative methods used to perform cognitive and behavioral tasks while simultaneously acquiring physiological data such as EMG, single-unit neuronal activity and/or local field potentials. Neuroscience Functional Mapping with Simultaneous MEG and EEG Hesheng Liu1, Naoaki Tanaka1, Steven Stufflebeam1, Seppo Ahlfors1, Matti Hämäläinen1 1Athinoula A. Martinos Center for Biomedical Imaging, MGH - Massachusetts General Hospital We use magnetoencephalography (MEG) and electroencephalography (EEG) to map brain areas involved in the processing of simple sensory stimuli. Biology Microsurgical Clip Obliteration of Middle Cerebral Aneurysm Using Intraoperative Flow Assessment Bob S. Carter1, Christopher Farrell1, Christopher Owen1 1Department of Neurosurgery , Havard Medical School, Massachusetts General Hospital Description of the surgical obliteration of a cerebral aneurysm utilizing an ultrasonic flow probe to assess arterial flow prior to and after aneurysm clip placement. Biology Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila Claudio Vinegoni1, Daniel Razansky2, Chrysoula Pitsouli3, Norbert Perrimon3, Vasilis Ntziachristos2, Ralph Weissleder1 1Center for Systems Biology, Massachusetts General Hospital, 2Institute for Biological and Medical Imaging (IBMI), Technical University of Munich and Helmholtz Center Munich, 3Department of Genetics, Harvard Medical School and Howard Hughes Medical Institute Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster. Biology Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging Claudio Vinegoni1,2, Daniel Razansky3, Jose-Luiz Figueiredo1,2, Lyuba Fexon1,2, Misha Pivovarov1,2, Matthias Nahrendorf1,2, Vasilis Ntziachristos3, Ralph Weissleder1,2 1Center for Systems Biology, Harvard Medical School, 2Center for Systems Biology, MGH - Massachusetts General Hospital, 3Institute for Biological and Medical Imaging, Technical University of Munich and Helmholtz Center Munich We suggest a Born normalized approach for Optical Projection Tomography (BnOPT) that accounts for the absorption properties of imaged samples to obtain accurate and quantitative fluorescence tomographic reconstructions. We use the proposed algorithm to reconstruct the fluorescence molecular probe distribution within small animal organs. Biology Homing of Hematopoietic Cells to the Bone Marrow Rushdia Z. Yusuf1, David T. Scadden1 1Center for Regenerative Medicine, MGH - Massachusetts General Hospital This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow. Biology Development of New Therapeutic Applications Using Microfluidics Mehmet Toner1 1Havard Medical School, MGH - Massachusetts General Hospital Biology Cell Capture Using a Microfluidic Device Kenneth Kotz1, Xuanhong Cheng1, Mehmet Toner1 1Havard Medical School, MGH - Massachusetts General Hospital Biology PDMS Device Fabrication and Surface Modification Kenneth Kotz1, Xuanhong Cheng1, Mehmet Toner1 1Havard Medical School, MGH - Massachusetts General Hospital Biology Organotypic Culture of Adult Rabbit Retina Ming H. Lye1, Tatjana C. Jakobs1, Richard H. Masland1, Amane Koizumi1 1Havard Medical School, MGH - Massachusetts General Hospital This article demonstrates the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells. Biology Propagation of Human Embryonic Stem (ES) Cells Laurence Daheron1 1Center for Regenerative Medicine, MGH - Massachusetts General Hospital
