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. Moses^{1,2 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 Herold^{1 1}Department 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 Mazor^2,3, Stephen X. Zhang^{3 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 Ding^1,2, Zhi-Qiang Lin^1,2, Jian-Ming Jiang^3,4, Christine E. Seidman^3,4, Jonathan G. Seidman^3,4, William T. Pu^1,2, Da-Zhi Wang^{1,2 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 Liu^2,3, Ping Zhu⁴, Zhongrong Li¹, Xue Li^{2,3 1}Department 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. Hagedorn^1,2, Jennifer L. Cillis³, Caitlyn R. Curley³, Taylor C. Patch³, Brian Li^1,2, Bradley W. Blaser^1,2,7, Raquel Riquelme^1,2, Leonard I. Zon^1,2,4,5,6, Dhvanit I. Shah^{1,2,3,4,5 1}Division 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. Surana^{1 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 Sanders^{4 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 Behar^1,4, Maria F. Pazyra-Murphy¹, Liliana Goumnerova⁵, Keith L. Ligon⁶, Charles D. Stiles¹, Rosalind A. Segal^{1 1}Department 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 Walch^{2 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. Pu^{1,2 1}Department 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. Walsh^3,4, Maria K. Lehtinen^{5 1}Department 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. Davies^1,2, Christine M. Barbon¹, Annie R. Voskertchian¹, Lee M. Nadler^1,2, Eva C. Guinan^{3,4 1}Medical 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. Pugach^1,2, Pulin Li^1,2, Richard White^1,2,3, Leonard Zon^{1,2 1}Department 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. Raschle^1,2, Michelle Lee¹, Roman Buechler¹, Joanna A. Christodoulou³, Maria Chang¹, Monica Vakil¹, Patrice L. Stering¹, Nadine Gaab^{1,3,4 1}Department 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. Cowan^{2 1}Department 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. Rosen^1,2, Michael F. Sweeney¹, John D. Mably^{1,2 1}Department 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.