S'identifier

Massachusetts General Hospital

76 ARTICLES PUBLISHED IN JoVE

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Biology

Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging
Claudio Vinegoni 1,2, Daniel Razansky 3, Jose-Luiz Figueiredo 1,2, Lyuba Fexon 1,2, Misha Pivovarov 1,2, Matthias Nahrendorf 1,2, Vasilis Ntziachristos 3, Ralph Weissleder 1,2
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.

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Biology

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila
Claudio Vinegoni 1, Daniel Razansky 2, Chrysoula Pitsouli 3, Norbert Perrimon 3, Vasilis Ntziachristos 2, Ralph Weissleder 1
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.

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Biology

Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury
Chiara Cianciolo Cosentino 1, Beth L. Roman 2, Iain A. Drummond 3, Neil A. Hukriede 1
1Department of Developmental Biology, University of Pittsburgh, 2Department of Biological Sciences, University of Pittsburgh, 3Department of Medicine and Genetics, Harvard Medical School

We describe a technique of microinjecting the aminoglycoside, gentamicin, into 2 days post-fetilization (dpf) zebrafish larvae to induce acute kidney injury (AKI). We also describe a method for whole mount immunohistochemistry, plastic embedding and sectioning of zebrafish larvae to visualize the AKI mediated damage.

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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
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.

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Bioengineering

Decellularization and Recellularization of Whole Livers
Basak E. Uygun 1, Gavrielle Price 1, Nima Saeidi 1, Maria-Louisa Izamis 1, Tim Berendsen 1, Martin Yarmush 1, Korkut Uygun 1
1Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Shriners Hospitals for Children

Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.

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Biology

Transplantation of Cells Directly into the Kidney of Adult Zebrafish
Cuong Q. Diep 1, Alan J. Davidson 1
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.

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Biology

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish
Jessica S. Blackburn 1, Sali Liu 1, David M. Langenau 2
1Department of Molecular Pathology, Massachusetts General Hospital, Harvard Medical School, 2Department of Molecular Pathology, Massachusetts General Hospital Cancer Center, Harvard Stem Cell Institute

Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.

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Medicine

How to Measure Cortical Folding from MR Images: a Step-by-Step Tutorial to Compute Local Gyrification Index
Marie Schaer 1, Meritxell Bach Cuadra 2,3, Nick Schmansky 4, Bruce Fischl 4, Jean-Philippe Thiran 2, Stephan Eliez 1
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.

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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. Applegate 4, 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
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.

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Biology

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning
Jamie Freeman 1, Janos Kriston-Vizi 1, Brian Seed 2, Robin Ketteler 1
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.

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JoVE Journal

Quantitative FRET (Förster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination
Yan Liu 1, Jiayu Liao 1
1Department of Bioengineering, Bourns College of Engineering, University of California, Riverside

A novel method involving quantitative analysis of FRET (Förster Resonance Energy Transfer) signals is described for studying enzyme kinetics. KM and kcat were obtained for the hydrolysis of the catalytic domain of SENP1 (SUMO/Sentrin specific protease 1) to pre-SUMO1 (Small Ubiquitin-like MOdifier). The general principles of this quantitative-FRET-based protease kinetic study can be applied to other proteases.

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Immunology and Infection

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals
Mathieu Angin 1, Melanie King 1, Marylyn Martina Addo 1,2
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.

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Biology

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay
Jessica M. Baker 1, Frederick M. Boyce 1
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.

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Biology

Assessing Murine Resistance Artery Function Using Pressure Myography
Mohd Shahid 1, Emmanuel S. Buys 1
1Anesthesia Center for Critical Care Research, Department of Anesthesia, Critical Care, and Pain Medicine, Massachusetts General Hospital, Harvard Medical School

In pressure myography, an intact small segment of a vessel is mounted onto two small cannulas and pressurized to a suitable luminal pressure. Here, we describe the method to measure vasorelaxation response of the mouse 3rd order mesenteric arteries in c57 and sGCα1-/- mice using pressure myography.

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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. Normandin 5, Cristian C. Constantinescu 6, Kelly P. Cosgrove 1,2,3
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.

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Biology

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs
Judith A. Sharp 1,2, Mike D. Blower 1,2
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.

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Biology

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy
Lisa Gfrerer 1, Max Dougherty 1, Eric C. Liao 1
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.

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Chemistry

Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications
Carlos Tassa 1, Monty Liong 1, Scott Hilderbrand 1, Jason E. Sandler 1, Thomas Reiner 1, Edmund J. Keliher 1, Ralph Weissleder 1, Stanley Y. Shaw 1
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.

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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 Pirzai 3, Bryan P. Hurley 1,3
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.

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Bioengineering

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood
Caroline N. Jones *1,2,3, Anh N. Hoang *1,2,3, Laurie Dimisko 1, Bashar Hamza 1, Joseph Martel 1,4, Daniel Irimia 1,2,3
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.

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Developmental Biology

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury
Caramai N. Kamei 1, Yan Liu 1,2, Iain A. Drummond 1,3
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.

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Behavior

Performing Behavioral Tasks in Subjects with Intracranial Electrodes
Matthew A. Johnson 1, Susan Thompson 1, Jorge Gonzalez-Martinez 2, Hyun-Joo Park 1, Juan Bulacio 2, Imad Najm 2, Kevin Kahn 4, Matthew Kerr 4, Sridevi V. Sarma 4, John T. Gale 1,3
1Department of Neurosciences, Cleveland Clinic Foundation, 2Epilepsy Center, Cleveland Clinic Foundation, 3Department of Neurosciences and Center for Neurological Restoration, Cleveland Clinic Foundation, 4Department of Biomedical Engineering, Johns Hopkins University

Patients implanted with intracranial electrodes provide a unique opportunity to record neurological data from multiple areas of the brain while the patient performs behavioral tasks. Here, we present a method of recording from implanted patients that can be reproducible at other institutions with access to this patient population.

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Behavior

Protocol for Studying Extinction of Conditioned Fear in Naturally Cycling Female Rats
Lisa Y. Maeng 1,2, Kara K. Cover 1, Aaron J. Landau 1, Mohammed R. Milad 1,2, Kelimer Lebron-Milad 1,2
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.

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Bioengineering

Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging
Thomas Reiner *1, Jason S. Lewis 1, Brian M. Zeglis *1
1Department of Radiology, Memorial Sloan Kettering Cancer Center

The bioorthogonal inverse electron demand Diels-Alder cycloaddition has been harnessed to create an effective and modular pretargeted PET imaging strategy for cancer. In this protocol, the steps of this methodology are described in the context of a model system employing the colorectal cancer targeted antibody huA33 and a 64Cu-labeled radioligand.

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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
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.

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Medicine

Ex Situ Normothermic Machine Perfusion of Donor Livers
Negin Karimian 1,2, Alix P.M. Matton 1,2, Andrie C. Westerkamp 1,2, Laura C. Burlage 1,2, Sanna op den Dries 1,2, Henri G.D. Leuvenink 2, Ton Lisman 1,2, Korkut Uygun 3, James F. Markmann 4, Robert J. Porte 1,2
1Section of Hepato-Pancreato-Biliary Surgery and Liver Transplantation, University of Groningen, University Medical Center Groningen, 2Surgical Research Laboratory, Department of Surgery, University of Groningen, University Medical Center Groningen, 3Center of Engineering in Medicine/Surgical Services, Massachusetts General Hospital, Harvard Medical School, and Shriners Burns Hospital, 4Division of Transplantation, Department of Surgery, Massachusetts General Hospital, Harvard Medical School

Here we present a protocol describing oxygenated ex situ machine perfusion of donor liver grafts. This article contains a step by step protocol to procure and prepare the liver graft for machine perfusion, prepare the perfusion fluid, prime the perfusion machine and perform oxygenated normothermic machine perfusion of the liver graft.

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Medicine

Functional Human Liver Preservation and Recovery by Means of Subnormothermic Machine Perfusion
Bote G. Bruinsma *1, James H. Avruch *2, Pepijn D. Weeder 1, Gautham V. Sridharan 1, Basak E. Uygun 1, Negin G. Karimian 1, Robert J. Porte 3, James F. Markmann 2, Heidi Yeh 2, Korkut Uygun 1
1Center for Engineering in Medicine, Dept. of Surgery, Massachusetts General Hospital, Harvard Medical School, 2Transplant Center, Dept. of Surgery, Massachusetts General Hospital, Harvard Medical School, 3Section of Hepatobiliary Surgery and Liver Transplantation, Department of Surgery, University Medical Center Groningen, University of Groningen

We describe a method of ex vivo machine perfusion of human liver grafts at subnormothermic temperature (21 °C).

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Medicine

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms
Manav Korpal 1, Jacob Feala 1, Xiaoling Puyang 1, Jian Zou 1, Alex H. Ramos 2, Jeremy Wu 1, Timm Baumeister 1, Lihua Yu 1, Markus Warmuth 1, Ping Zhu 1
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.

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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 Kong 1, Eric C. Liao 1
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.

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JoVE Core

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite
Vishal Keshari 1, Basma Adeeb 1, Alison E. Simmons 1, Thomas W. Simmons 1, Cuong Q. Diep 1
1Department of Biology, Indiana University of Pennsylvania

Exposure to teratogens may cause birth defects. Zebrafish are useful for determining the teratogenic potential of chemicals. We demonstrate the utility of zebrafish by exposing embryos to various levels of nitrite and also at different times of exposure. We show that nitrite can be toxic and cause severe developmental defects.

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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-Gabrieli 4, Catherine J. Chu 1,5, Alvaro Pascual-Leone 1,2,3, Bernard S. Chang 1,2
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.

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Biology

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization
Melis N. Anahtar 1, Brittany A. Bowman 1, Douglas S. Kwon 1
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.

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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. Burke 2, Trejeeve Martyn 1, Timothy E. Thayer 2, Hannah R. Shakartzi 1, Mary D. Buswell 1, Robert E. Tainsh 1, Binglan Yu 1,4, Aranya Bagchi 1,4, David K. Rhee 2,4, Connie Wu 1,2,4, Matthias Derwall 5, 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
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, 3Cardiovascular Division, Brigham and Women's Hospital, 4Harvard Medical School, 5Department of Anesthesiology, Uniklinik RWTH Aachen, RWTH Aachen University, 6Center for Immunology and Inflammatory Diseases and the Division of Rheumatology, Allergy, and Immunology of the Department of Medicine, Massachusetts General Hospital

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.

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Bioengineering

A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting
Gabriel E. Büchel 1,2, Brandon Carney 1,3, Jun Tang 1, Brian M. Zeglis 1,3, Jörg Eppinger 2, Thomas Reiner 1,4
1Department of Radiology, Memorial Sloan Kettering Cancer Center, 2KAUST Catalysis Center, King Abdullah University of Science and Technology, 3Department of Chemistry, Hunter College, and PhD Program in Chemistry, Graduate Center of City University of New York, 4Department of Radiology, Weill Cornell Medical College

This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.

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Cancer Research

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines
Jun Tang 1, Carlos Pérez-Medina 1,2, Yiming Zhao 2, Ahmad Sadique 1, Willem J. M. Mulder 2, Thomas Reiner 1
1Department of Radiology, Memorial Sloan Kettering Cancer Center, 2Translational and Molecular Imaging Institute, Icahn School of Medicine at Mount Sinai

The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

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Developmental Biology

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae
Felix Ellett 1, Daniel Irimia 1
1BioMEMS Resource Center, Department of Surgery, Massachusetts General Hospital–Harvard Medical School–Shriners Burns Hospital

Microinjection of zebrafish embryos and larvae is a crucial but challenging technique used in many zebrafish models. Here, we present a range of microscale tools to aid in the stabilization and orientation of zebrafish for both microinjection and imaging.

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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 Gutu 2, Alexander D. Eaton 1, Amy L. Silverio 1, Frederick M. Ausubel 2,3, Bryan P. Hurley 1,4, Lael M. Yonker 1,4
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.

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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
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.

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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
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.

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Medicine

Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy
Eriko Katsuta 1, Masanori Oshi 1, Omar M. Rashid 2,3,4,5, Kazuaki Takabe 1,6,7,8,9,10
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.

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Medicine

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice
Caryn Porter 1, Maria Ennamorati 2, Nitya Jain 2
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.

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JoVE Journal

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-Byrne 1, Angela Tung 1, James J. Collins 1,4,5,6,7, George M. Church 1,2
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.

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JoVE Journal

Chemoselective Preparation of 1-Iodoalkynes, 1,2-Diiodoalkenes, and 1,1,2-Triiodoalkenes Based on the Oxidative Iodination of Terminal Alkynes
Youzhi Li *1, Daya Huang *1, Ju Huang 1, Yan Liu 1, Keiji Maruoka 1,2
1School of Chemical Engineering and Light Industry, Guangdong University of Technology, 2Department of Chemistry, Graduate School of Science, Kyoto University

Herein, detailed protocols for the oxidative iodination of terminal alkynes using hypervalent-iodine reagents are presented, which chemoselectively afford 1-iodoalkynes, 1,2-diiodoalkenes, and 1,1,2-triiodoalkenes.

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Bioengineering

Apparatus for Harvesting Tissue Microcolumns
Joshua Tam 1,2, William Farinelli 1, Walfre Franco 1,2, R. Rox Anderson 1,2
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.

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Developmental Biology

Generation of Human Primordial Germ Cell-like Cells at the Surface of Embryoid Bodies from Primed-pluripotency Induced Pluripotent Stem Cells
Shino Mitsunaga 1, Keiko Shioda 1, Kurt J. Isselbacher 1, Jacob H. Hanna 2, Toshi Shioda 1
1Center for Cancer Research, Massachusetts General Hospital, 2Department of Molecular Genetics, Weizmann Institute of Science

Primordial germ cells (PGCs) are common precursors of both sperm and eggs. Human embryonic PGCs are specified from pluripotent epiblast cells through interactions of cytokines. Here, we describe a 13-day protocol of inducing human cells transcriptomally resembling PGCs at the surface of embryoid bodies from primed-pluripotency induced pluripotent stem cells.

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Behavior

A Behavioral Test Battery for the Repeated Assessment of Motor Skills, Mood, and Cognition in Mice
Ran You 1,2,3, Yan Liu 3, Raymond Chuen-Chung Chang 3
1Nanjing Key Laboratory of Pediatrics, Children's Hospital of Nanjing Medical University, 2Nanjing Key Laboratory of Pediatrics, Nanjing Medical University, 3Laboratory of Neurodegenerative Disease, Li Ka Shing Faculty of Medicine, University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region (HK SAR)

A comprehensive behavioral test battery of motor skills, mood—including social interaction, depression, and anxiety—and cognition is designed for the repeated assessment of neurodegeneration-related behavioral changes in mice.

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Neuroscience

A Protocol for Transcranial Photobiomodulation Therapy in Mice
Farzad Salehpour 1,2, Luis De Taboada 3, Paolo Cassano 4,5,6, Farzin Kamari 1, Javad Mahmoudi 1, Sohrab Ahmadi-Kandjani 7, Seyed Hossein Rasta 8,9,10, Saeed Sadigh-Eteghad 1
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.

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JoVE Core

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
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.

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Bioengineering

Bulk Droplet Vitrification for Primary Hepatocyte Preservation
Reinier J. de Vries 1,2,3, Peony D. Banik 1,2, Sonal Nagpal 1,2, Lindong Weng 1,2, Sinan Ozer 1,2, Thomas M. van Gulik 3, Mehmet Toner 1,2, Shannon N. Tessier 1,2, Korkut Uygun 1,2
1Center for Engineering in Medicine, Harvard Medical School, Massachusetts General Hospital, 2Shriners Hospitals for Children, Boston, 3Department of Surgery, University of Amsterdam

This manuscript describes an ice-free cryopreservation method for large quantities of rat hepatocytes whereby primary cells are pre-incubated with cryoprotective agents at a low concentration and vitrified in large droplets.

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Neuroscience

Real-Time fMRI Brain Mapping in Animals
Sangcheon Choi 1,2, Kengo Takahashi 1,2, Yuanyuan Jiang 1,3, Sascha Köhler 4, Hang Zeng 1,2, Qi Wang 1,2, Yan Ma 1,2, Xin Yu 1,3
1Max Planck Institute for Biological Cybernetics, 2Graduate Training Centre of Neuroscience, 3MGH/MIT/HMS Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Harvard Medical School, Massachusetts General Hospital, 4Bruker BioSpin

Animal brain functional mapping can benefit from the real-time functional magnetic resonance imaging (fMRI) experimental set-up. Using the latest software implemented in the animal MRI system, we established a real-time monitoring platform for small animal fMRI.

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JoVE Journal

A Model Membrane Platform for Reconstituting Mitochondrial Membrane Dynamics
Yifan Ge 1, Sivakumar Boopathy 1, Adam Smith 2, Luke H. Chao 1,3
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.

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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
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.

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Medicine

A Novel Inhalation Mask System to Deliver High Concentrations of Nitric Oxide Gas in Spontaneously Breathing Subjects
Riccardo Pinciroli 1, Lisa Traeger 1, Anna Fischbach 1, Stefano Gianni 1, Caio Cesar Araujo Morais 1, Bijan Safaee Fakhr 1, Raffaele Di Fenza 1, Dianna Robinson 3, Ryan Carroll 2, Warren M. Zapol 1, Lorenzo Berra 1
1Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital and Harvard Medical School, 2Division of Pediatric Critical Care Medicine, Department of Pediatrics, Massachusetts General Hospital and Harvard Medical School, 3Respiratory Care, Massachusetts General Hospital

This simple and highly adaptable system device for the inhalation of high-concentration nitric oxide (NO) gas does not require mechanical ventilators, positive pressure, or high gas flows. Standard medical consumables and a snug-fitting mask are used to safely deliver NO gas to spontaneously breathing subjects.

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Biology

Partial Hepatectomy in Adult Zebrafish
Isaac M. Oderberg 1,2, Wolfram Goessling 1,2,3
1Harvard Medical School, 2Brigham and Women’s Hospital, 3Massachusetts General Hospital

This protocol describes the procedure for removing the ventral lobe of the liver in adult zebrafish to enable the study of liver regeneration.

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Developmental Biology

A Simplified Method for Generating Kidney Organoids from Human Pluripotent Stem Cells
Aneta Przepiorski 1, Amanda E. Crunk 1, Teresa M. Holm 2, Veronika Sander 2, Alan J. Davidson 2, Neil A. Hukriede 1,3
1Department of Developmental Biology, University of Pittsburgh, School of Medicine, 2Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, 3Center for Critical Care Nephrology, University of Pittsburgh, School of Medicine

Here we describe a protocol to generate kidney organoids from human pluripotent stem cells (hPSCs). This protocol generates kidney organoids within two weeks. The resulting kidney organoids can be cultured in large-scale spinner flasks or multi-well magnetic stir plates for parallel drug-testing approaches.

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Neuroscience

Co-Culturing Microglia and Cortical Neurons Differentiated from Human Induced Pluripotent Stem Cells
Kara Lopez-Lengowski 1,2, Annie Kathuria 1,2,3, Kaia Gerlovin 1,2, Rakesh Karmacharya 1,2,3,4,5,6,7
1Center for Genomic Medicine, Massachusetts General Hospital, 2Chemical Biology Program, Broad Institute of MIT & Harvard, 3Department of Psychiatry, Harvard Medical School, 4Schizophrenia and Bipolar Disorder Program, McLean Hospital, 5Program in Neuroscience, Harvard University, 6Program in Chemical Biology, Harvard University, 7Harvard Stem Cell Institute

This protocol describes a methodology to differentiate microglia from human iPSCs and maintain them in co-culture with iPSC-derived cortical neurons in order to study mechanistic underpinnings of neuroimmune interactions using human neurons and microglia.

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Immunology and Infection

Partial Heterotopic Hindlimb Transplantation Model in Rats
Marion Goutard 1,2,3, Mark A. Randolph 1,2,3, Corentin B. Taveau 1,2,3,4, Elise Lupon 1,2,3, Laurent Lantieri 4, Korkut Uygun 1,2,3, Curtis L. Cetrulo Jr. *1,2,3, Alexandre G. Lellouch *1,2,3,4
1Division of Plastic and Reconstructive Surgery, Massachusetts General Hospital, Harvard Medical School, 2Vascularized Composite Allotransplantation Laboratory, Center for Transplantation Sciences, Massachusetts General Hospital, Harvard Medical School, 3Shriners Hospital for Children, 4Service de Chirurgie Plastique, Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris (APHP), Université de Paris

This paper presents a partial heterotopic osteomyocutaneous flap transplantation protocol in rats and its potential outcomes in the mid-term follow-up.

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Medicine

Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome
Jacob Vieira 1, Tara Gallagher 2, Hui-Yu Sui 1, Sirus Jesudasen 3, Katrine Whiteson 2, George A. O'Toole 4, Kurt Hanselmann 5, Peggy S. Lai 1
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.

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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
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.

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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
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.

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Bioengineering

A Reliable Porcine Fascio-Cutaneous Flap Model for Vascularized Composite Allografts Bioengineering Studies
Victor Pozzo 1,2,4, Golda Romano 1,2,4, Marion Goutard 1,2,4, Elise Lupon 1,2,4, Pierre Tawa 1,2,4, Aylin Acun 3,4,5, Alec R. Andrews 2, Corentin B. Taveau 1,2,4,6, Basak E. Uygun 1,2,3,4, Mark A. Randolph 1,2,4, Curtis L. Cetrulo 1,2,4, Alexandre G. Lellouch 1,2,4,6
1Division of Plastic and Reconstructive Surgery, Massachusetts General Hospital, Harvard Medical School, 2Vascularized Composite Allotransplantation Laboratory, Center for Transplantation Sciences, Massachusetts General Hospital, Harvard Medical School, 3Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, Harvard Medical School, 4Shriners Hospital for Children, 5Department of Biomedical Engineering, Widener University, 6Service de Chirurgie Plastique, Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris (APHP), Université Paris Descartes

The present protocol describes the porcine fascio-cutaneous flap model and its potential use in vascularized composite tissue research.

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Medicine

Point-Of-Care Ultrasound Screening for Proximal Lower Extremity Deep Venous Thrombosis
Rebecca G. Theophanous 1, Vinca W. Chow 2, David L. Convissar 3, Stephen C. Haskins 4,5, Robert A. Jones 6, Hari K. P. Kalagara 7, Yuriy S. Bronshteyn 8
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.

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Neuroscience

Use of a Foot-Induced Digitally Controlled Resistance Device for Functional Magnetic Resonance Imaging Evaluation in Patients with Foot Paresis
Mark P. Ottensmeyer 1,2, Sabrina Elbach 3,4, Loukas Astrakas 3,5, Shasha Li 2,3,4, A. Aria Tzika 2,3,4
1Medical Device & Simulation Laboratory, Department of Radiology, Massachusetts General Hospital, 2Harvard Medical School, Harvard University, 3NMR Surgical Laboratory, Department of Surgery, Shriners Children’s Boston, Massachusetts General Hospital, 4Athinoula A. Martinos Center of Biomedical Imaging, Department of Radiology, Massachusetts General Hospital, 5Medical Physics Laboratory, Faculty of Medicine, University of Ioannina

Chronic stroke patients' insured rehabilitation is generally time limited. Imaging-based study of brain activity from walking-related motor tasks can lead to establishing biomarkers to measure improved outcomes and justify extending tailored therapy. A novel, magnetic resonance-compatible, variable-resistance foot motion device and a protocol for use during functional magnetic resonance imaging are presented.

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Immunology and Infection

A Novel Method to Determine the Longitudinal Antibacterial Activity of Drug-Eluting Materials
Amita Sekar 1,2, Sashank Lekkala 1, Ebru Oral 1,2
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.

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JoVE Core

Point-of-Care Lung Ultrasound in Adults: Image Acquisition
Rodrigo O. L. Pereira 1, David L. Convissar 2, Sean Montgomery 3, James T. Herbert 1, Christopher R. Reed 3, Hoang J. Tang 3, Yuriy S. Bronshteyn 1,4
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.

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Medicine

Image Acquisition Method for the Sonographic Assessment of the Inferior Vena Cava
Maximilian Hoffman 1, David L. Convissar 2, Marie-Louise Meng 1, Sean Montgomery 4, Yuriy S. Bronshteyn 1,3
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.

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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 Bertheuil 3, 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
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, 5Shriners Children's Boston, 6Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, 7Department of Plastic Surgery and Hand Surgery, University Medical Center Utrecht, 8Department of Plastic, Reconstructive, and Aesthetic Surgery, Groupe Almaviva Santé, Clinique de l'Alma

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.

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Medicine

Focused Assessment with Sonography for Trauma (FAST) Exam: Image Acquisition
John D. Ritchie 1, Charles N. Trujillo 2, David L. Convissar 3, William Shihao Lao 4, Sean Montgomery 5, Yuriy S. Bronshteyn 6
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.

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Neuroscience

Manual Segmentation of the Human Choroid Plexus Using Brain MRI
Deepthi Bannai *1,2, Yuan Cao *3,4, Matcheri Keshavan 1,2, Martin Reuter 5,6,7, Paulo Lizano 1,2,8
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.

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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
1Vascularized Composite Allotransplantation Laboratory, Massachusetts General Hospital, 2Harvard Medical School, 3Shriners Children’s Boston, 4Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, 5Department of Vascular Surgery, Centre Hospitalier Universitaire Vaudois and University of Lausanne, 6Department of Plastic, Reconstructive and Hand Surgery, University Medical Center Utrecht, 7Department of Plastic, Reconstructive and Aesthetic Surgery, Rennes University Hospital Center (CHU de Rennes), Rennes 1 University, 8Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, 9Plastic Surgery Research Laboratory, Massachusetts General Hospital

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.

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Medicine

Gastric Point of Care Ultrasound in Adults: Image Acquisition and Interpretation
Eric R. Heinz 1, Omar Al-Qudsi 2, David L. Convissar 3, Marianne D. David 1, Jennifer E. Dominguez 2, Stephen Haskins 4, Christina Jelly 5, Anahi Perlas 6, Anita N. Vincent 1, Yuriy S. Bronshteyn 2
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.

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Immunology and Infection

Stromal Cell Isolation From Hematopoietic Organs
Trine Kristiansen *1,2,3, Christina Mayerhofer *1,2,3, Karin Gustafsson 1,2,3, David T. Scadden 1,2,3
1Center for Regenerative Medicine, Massachusetts General Hospital, 2Harvard Stem Cell Institute, 3Department of Stem Cell and Regenerative Biology, Harvard University

Here we present protocols that enable isolation of stromal cells from murine bone, bone marrow, thymus and human thymic tissue compatible with single-cell multiomics.

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Immunology and Infection

Epithelial Cell Infection Analyses with Shigella
Kender Poore *1, Bryan R. Lenneman *1,2, Christina S. Faherty 1,2
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.

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Cancer Research

Dissociation of Human and Mouse Tumor Tissue Samples for Single-cell RNA Sequencing
Jacy Fang *1,2, Isabella Salinas *1,2, Sarah San Vicente *1,2, Caroline Zielinski *1,2, Moshe Sade-Feldman 1,2,3
1Krantz Family Center for Cancer Research, Department of Medicine, Massachusetts General Hospital, 2Broad Institute of MIT and Harvard, 3Department of Medicine, Harvard Medical School

This protocol outlines the procedure for rapidly dissociating human and mouse tumor samples for single-cell RNA sequencing.

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Bioengineering

Modified Langendorff Perfusion for Extended Perfusion Times of Rodent Cardiac Grafts
Casie A. Pendexter 1,2, Maya Bolger-Chen 1,2, Manuela Lopera Higuita 1,2, Stephanie E. J. Cronin 1,2, S. Alireza Rabi 3, Asishana A. Osho 3, Shannon N. Tessier 1,2
1Center for Engineering in Medicine and Surgery, Harvard Medical School and Massachusetts General Hospital, 2Shriners Children's Boston, 3Division of Cardiac Surgery, Corrigan Minehan Heart Center, Massachusetts General Hospital

This article demonstrates the feasibility of achieving longer perfusion times (4 h) of murine cardiac grafts without function loss by employing lower (30-35 mmHg) than physiological (60-80 mmHg) perfusion pressures during Langendorff.

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Immunology and Infection

Identification of Rare Antigen-Specific T Cells from Mouse Lungs with Peptide:Major Histocompatibility Complex Tetramers
Daniel S. Shin 1,2,3, Juliana Barreto de Albuquerque 1,3, James J. Moon 1,3,4
1Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy, and Immunology, Massachusetts General Hospital, 2Division of Immunology, Boston Children's Hospital, 3Harvard Medical School, 4Division of Pulmonary and Critical Care Medicine, Massachusetts General Hospital

We provide a detailed protocol for isolating and identifying rare antigen-specific T cell populations in mouse lungs through magnetic bead-based T cell enrichment and peptide:major histocompatibility complex (MHC) tetramers.

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