Massachusetts General Hospital

54 articles published in JoVE

• 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. Bronshteyn^{6 1}Trauma, 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

  
  Modified Tail Vein and Penile Vein Puncture for Blood Sampling in the Rat Model Laura Charlès^1,2,3, Thomas Agius*^4,5, Irina Filz von Reiterdank*^1,2,3,6, Janna Hagedorn^1,2,3, Yanis Berkane^1,2,3,7, Hyshem H. Lancia^1,2,3, Basak E. Uygun^2,3,8, Korkut Uygun^2,3,8, Curtis L. Cetrulo Jr.^1,2,3,9, Mark A. Randolph^1,2,3,9, Alexandre G. Lellouch^{1,2,3,8 1}Vascularized 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. Bronshteyn^{1,4 1}Department 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 Sekar^1,2, Sashank Lekkala¹, Ebru Oral^{1,2 1}Harris 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. Haskins^4,5, Robert A. Jones⁶, Hari K. P. Kalagara⁷, Yuriy S. Bronshteyn^{8 1}Department 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 Reyes^1,2, Hyshem H. Lancia^1,2,5, Irina Filz von Reiterdank^1,2,5,6,7, Nicolas Bertheuil³, Basak E. Uygun^2,5,6, Korkut Uygun^1,2,5,6, William G. Austen Jr.^2,4, Curtis L. Cetrulo Jr.^1,2,4,5, Mark A. Randolph^1,2,4,5, Alexandre G. Lellouch^{1,2,5,6,8 1}Vascularized 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. Bronshteyn^{1,3 1}Department 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 Subramanian^{1,2 1}Molecular 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 Fenza^1,3, Binglan Yu^1,3, Ryan W. Carroll^2,3, Lorenzo Berra^{1,3 1}Department 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. Lai^{1 1}Division 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. Oderberg^1,2, Wolfram Goessling^{1,2,3 1}Harvard 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 Pong^1,2,3, Paulo Lizano^1,2,3,4, Rakesh Karmacharya^{1,3,4,5 1}Center 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. Chao^{1,3 1}Department 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. Ottensmeyer^1,2, Shasha Li^2,3,4, Gianluca De Novi^1,2, A. Aria Tzika^{2,3,4 1}Medical 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. Rashid^2,3,4,5, Kazuaki Takabe^{1,6,7,8,9,10 1}Breast 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 Salehpour^1,2, Luis De Taboada³, Paolo Cassano^4,5,6, Farzin Kamari¹, Javad Mahmoudi¹, Sohrab Ahmadi-Kandjani⁷, Seyed Hossein Rasta^8,9,10, Saeed Sadigh-Eteghad^{1 1}Neurosciences 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 Tam^1,2, William Farinelli¹, Walfre Franco^1,2, R. Rox Anderson^{1,2 1}Wellman 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 Yeo^1,2, Xiaoge Guo^1,2, Alissa Lance-Byrne¹, Angela Tung¹, James J. Collins^1,4,5,6,7, George M. Church^{1,2 1}Wyss 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 Jain^{2 1}Center 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. Nickerson^1,2, Christina S. Faherty^{1,2 1}Mucosal 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. Ausubel^2,3, Bryan P. Hurley^1,4, Lael M. Yonker^{1,4 1}Department 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. Adamala^1,3, Jack W. Szostak^{1 1}Howard 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. Shafi^1,2,3, Susan Whitfield-Gabrieli⁴, Catherine J. Chu^1,5, Alvaro Pascual-Leone^1,2,3, Bernard S. Chang^{1,2 1}Department 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. Hutcheson^3,4, Megan F. Burke², Trejeeve Martyn¹, Timothy E. Thayer², Hannah R. Shakartzi¹, Mary D. Buswell¹, Robert E. Tainsh¹, Binglan Yu^1,4, Aranya Bagchi^1,4, David K. Rhee^2,4, Connie Wu^1,2,4, Matthias Derwall⁵, Emmanuel S. Buys^1,4, Paul B. Yu^3,4, Kenneth D. Bloch^1,2,4, Elena Aikawa^3,4, Donald B. Bloch^1,5,6, Rajeev Malhotra^{2,4 1}Anesthesia 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. Kwon^{1 1}Ragon 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 Zhu^{1 1}H3 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 Liu^1,2, Iain A. Drummond^{1,3 1}Nephrology 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. Maeng^1,2, Kara K. Cover¹, Aaron J. Landau¹, Mohammed R. Milad^1,2, Kelimer Lebron-Milad^{1,2 1}Department 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. Moore^1,2, David M. Langenau^{1,2 1}Molecular 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 rag2^E450fs 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 Zhang^1,2, Chaincy Kuo³, Anna Moore¹, Chongzhao Ran^{1 1}Molecular 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. Tolonen^1,2,3, Wilhelm Haas^{4 1}CEA, 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 Martel^1,4, Daniel Irimia^{1,2,3 1}The 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. Boyce^{1 1}Department 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. Kusek^1,2,3, Michael A. Pazos^1,3, Waheed Pirzai³, Bryan P. Hurley^{1,3 1}Department 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. Liao^{1 1}Center 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. Shaw^{1 1}Center 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. Morris^1,2,3,4, Su Jin Kim^1,3, Jenna M. Sullivan^1,3,4, Shuo Wang^3,4, Marc D. Normandin⁵, Cristian C. Constantinescu⁶, Kelly P. Cosgrove^{1,2,3 1}Diagnostic 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. Sharp^1,2, Mike D. Blower^{1,2 1}Department 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 Addo^{1,2 1}Ragon 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. Hariri^1,2,3, Matthew B. Applegate⁴, Mari Mino-Kenudson^1,2, Eugene J. Mark^1,2, Brett E. Bouma^2,3, Guillermo J. Tearney^1,2,3, Melissa J. Suter^{2,3,5 1}Department 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 Ketteler^{1 1}MRC 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 Cuadra^2,3, Nick Schmansky⁴, Bruce Fischl⁴, Jean-Philippe Thiran², Stephan Eliez^{1 1}Department 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. Davidson^{1 1}Center 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. Sheth^1,2, Emad N. Eskandar^{1,2 1}Nayef 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 Hämäläinen^{1 1}Athinoula 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 Owen^{1 1}Department 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 Weissleder^{1 1}Center 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 Vinegoni^1,2, Daniel Razansky³, Jose-Luiz Figueiredo^1,2, Lyuba Fexon^1,2, Misha Pivovarov^1,2, Matthias Nahrendorf^1,2, Vasilis Ntziachristos³, Ralph Weissleder^{1,2 1}Center 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. Scadden^{1 1}Center 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 Toner^{1 1}Havard Medical School, MGH - Massachusetts General Hospital

• Biology

  
  Cell Capture Using a Microfluidic Device Kenneth Kotz¹, Xuanhong Cheng¹, Mehmet Toner^{1 1}Havard Medical School, MGH - Massachusetts General Hospital

• Biology

  
  PDMS Device Fabrication and Surface Modification Kenneth Kotz¹, Xuanhong Cheng¹, Mehmet Toner^{1 1}Havard Medical School, MGH - Massachusetts General Hospital

• Biology

  
  Organotypic Culture of Adult Rabbit Retina Ming H. Lye¹, Tatjana C. Jakobs¹, Richard H. Masland¹, Amane Koizumi^{1 1}Havard 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 Daheron^{1 1}Center for Regenerative Medicine, MGH - Massachusetts General Hospital