Children's Hospital Boston View Institution's Website 17 articles published in JoVE Cancer Research A Time-lapse, Label-free, Quantitative Phase Imaging Study of Dormant and Active Human Cancer Cells Jing Huang*1,2, Peng Guo*1,2, Marsha A. Moses1,2 1 Dormant and active cancer cell phenotypes were characterized using quantitative phase imaging. Cell proliferation, migration, and morphology assays were integrated and analyzed in one simple method. Medicine Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease Martin Wagner1, Claudia Baer1, Werner Zuschratter2, Monika Riek-Burchardt3, Christian Deffge1, Soenke Weinert1, Jerry C Lee4, Ruediger C Braun-Dullaeus1, Joerg Herold1 1Department of Cardiology and Angiology, University of Magdeburg, 2Leibniz Institute for Neurobiology, 3Institute of Molecular and Clinical Immunology, University of Magdeburg, 4 Monocytes are important mediators of arteriogenesis in the context of peripheral arterial disease. Using a basement membrane-like matrix and intravital microscopy, this protocol investigates monocyte homing and tumor-related angiogenesis after monocyte injection in the femoral artery ligation murine model. Neuroscience Measuring and Altering Mating Drive in Male Drosophila melanogaster Christine L. Boutros*1, Lauren E. Miner*1, Ofer Mazor2,3, Stephen X. Zhang3 1 This article describes a behavioral assay that uses male mating drive in Drosophila melanogaster to study motivation. Using this method, researchers can utilize advanced fly neurogenetic techniques to uncover the genetic, molecular, and cellular mechanisms that underlie this motivation. Genetics Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts Jian Ding1,2, Zhi-Qiang Lin1,2, Jian-Ming Jiang3,4, Christine E. Seidman3,4, Jonathan G. Seidman3,4, William T. Pu1,2, Da-Zhi Wang1,2 1 In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described. Developmental Biology Rapid Acquisition of 3D Images Using High-resolution Episcopic Microscopy Haochuan Zhang*1,2,3, JunGang Huang*2,3,4, Xin Liu2,3, Ping Zhu4, Zhongrong Li1, Xue Li2,3 1Department of Pediatric Surgery, The Second Affiliated Hospital & Yuying Children's Hospital, Wenzhou Medical University, 2 We describe a detailed protocol using high-resolution episcopic microscopy to acquire three-dimensional (3D) images of mouse embryos. This improved protocol utilizes a modified tissue preparation method to enhance penetration of the fluorescent dye, thereby permitting morphometric analysis of both small and large-sized specimens. Developmental Biology Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae Elliott J. Hagedorn1,2, Jennifer L. Cillis3, Caitlyn R. Curley3, Taylor C. Patch3, Brian Li1,2, Bradley W. Blaser1,2,7, Raquel Riquelme1,2, Leonard I. Zon1,2,4,5,6, Dhvanit I. Shah1,2,3,4,5 1Division of Hematology, Department of Medicine, Brigham and Women’s Hospital, 4Harvard Stem Cell Institute, 5Broad Institute of Massachusetts Institute of Technology, 6Howard Hughes Medical Institute, 7Division of Hematologic Malignancies, Dana-Farber Cancer Institute This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest. Immunology and Infection Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes Cheryn J. Couter1, Neeraj K. Surana1 1 There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization. Developmental Biology The Complete and Updated "Rotifer Polyculture Method" for Rearing First Feeding Zebrafish Christian Lawrence1, Jason Best1, Jason Cockington2, Eric C. Henry3, Shane Hurley1, Althea James1, Christopher Lapointe1, Kara Maloney1, Erik Sanders4 1 Larval zebrafish are adapted to feed on zooplankton. It is possible to capitalize on this natural feature in the laboratory by growing first feeding fish together in the same system with live saltwater rotifers. This "polyculture" strategy promotes high growth and survival with minimal labor and disturbance to the larvae. Medicine A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies Emily J. Chadwick1, David P. Yang1, Mariella G. Filbin2, Emanuele Mazzola3, Yu Sun1, Oded Behar1,4, Maria F. Pazyra-Murphy1, Liliana Goumnerova5, Keith L. Ligon6, Charles D. Stiles1, Rosalind A. Segal1 1Department of Cancer Biology, Dana-Farber Cancer Institute, 2 Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. Biology A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells Farokh Dotiwala1, Isabelle Fellay2, Luis Filgueira2, Denis Martinvalet3, Judy Lieberman1, Michael Walch2 1 We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease. Medicine Ultrasound-guided Transthoracic Intramyocardial Injection in Mice Terence W. Prendiville1, Qing Ma1, Zhiqiang Lin1, Pingzhu Zhou1, Aibin He1, William T. Pu1,2 1Department of Cardiology, Boston Children's Hospital, 2Harvard Stem Cell Institute, Harvard University Echocardiography-guided percutaneous intramyocardial injection represents an efficient, reliable, and targetable modality for the delivery of gene transfer agents or cells into the murine heart. Following the steps outlined in this protocol, the operator can quickly become competent in this versatile, minimally invasive technique. Neuroscience Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants Mauro W. Zappaterra1, Anthony S. LaMantia2, Christopher A. Walsh3,4, Maria K. Lehtinen5 1Department of Physical Medicine and Rehabilitation, VA Greater Los Angeles Healthcare System, 2Department of Pharmacology and Physiology, Institute for Neuroscience, The George Washington University School of Medicine and Health Sciences, 3Division of Genetics, Department of Medicine, Boston Children's Hospital, 4Howard Hughes Medical Institute, Boston Children's Hospital, 5Department of Pathology, Boston Children's Hospital, Harvard Medical School The ventricular cerebrospinal fluid (CSF) bathes the neuroepithelial and cerebral cortical progenitor cells during early brain development in the embryo. Here we describe the method developed to isolate ventricular CSF from rodent embryos of different ages in order to investigate its biological function. In addition, we demonstrate our cerebral cortical explant dissection and culture technique that allows for explant growth with minimal volumes of culture medium or CSF. Immunology and Infection Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade Jeff K. Davies1,2, Christine M. Barbon1, Annie R. Voskertchian1, Lee M. Nadler1,2, Eva C. Guinan3,4 1Medical Oncology, Dana Farber Cancer Institute, 2Department of Medicine, Brigham and Womens Hospital, 3Pediatric Oncology, Dana Farber Cancer Institute, 4Division of Hematology/Oncology, Children’s Hospital Boston This paper describes a simple technique to induce alloantigen-specific anergy in human peripheral blood mononuclear cells. The technique can be applied clinically to generate non-alloreactive donor cells. Infusion of these cells could improve immune reconstitution and reduce toxicity after allogeneic hematopoietic stem cell transplantation. Biology Retro-orbital Injection in Adult Zebrafish Emily K. Pugach1,2, Pulin Li1,2, Richard White1,2,3, Leonard Zon1,2 1Department of Hematology and Oncology, Children’s Hospital Boston, 2Harvard Stem Cell Institute, Harvard Medical School, 3Department of Medical Oncology, Dana Farber Cancer Institute Here we show how to do retro-orbital injection in adult zebrafish. Biology Making MR Imaging Child's Play - Pediatric Neuroimaging Protocol, Guidelines and Procedure Nora M. Raschle1,2, Michelle Lee1, Roman Buechler1, Joanna A. Christodoulou3, Maria Chang1, Monica Vakil1, Patrice L. Stering1, Nadine Gaab1,3,4 1Department of Developmental Medicine, Children’s Hospital Boston, 2Department of Neuropsychology, University of Zurich, 3Graduate School of Education, Harvard, 4Harvard Medical School Despite an increase in the use of structural and functional magnetic resonance imaging (fMRI) in humans, the study of young pediatric populations remains a challenge. We present a hands-on, step-by-step video protocol including guidelines for clinicians and researchers intending to perform (f)MRI in young children. Biology Implantation of Engineered Tissue in the Rat Heart Bjoern Sill1, Ivan V. Alpatov2, Christina A. Pacak2, Douglas B. Cowan2 1Department of Anesthesiology, Perioperative and Pain Medicine, Children's Hospital Boston and Harvard Medical School, 2Department of Anesthesiology, Perioperative and Pain Medicine, Children’s Hospital Boston Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat. Biology Microinjection of Zebrafish Embryos to Analyze Gene Function Jonathan N. Rosen1,2, Michael F. Sweeney1, John D. Mably1,2 1Department of Genetics, Harvard Medical School, 2Department of Cardiology, Children’s Hospital Boston This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.