27 articles published in JoVE

• Immunology and Infection

  
  Isolation of Leukocytes from Human Breast Milk for Use in an Antibody-dependent Cellular Phagocytosis Assay of HIV Targets Rebecca L.R. Powell¹, Alisa Fox^{1 1}Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai Breast milk transmits human immunodeficiency virus (HIV), though only ~15% of infants breastfed by HIV-infected mothers become infected. Breastfed infants ingest ~10⁵−10⁸ maternal leukocytes daily, though these cells are understudied. Here we describe the isolation of breast milk leukocytes and an analysis of their phagocytic capacity.

• Cancer Research

  
  Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts Dan Han¹, Veronica Rodriguez-Bravo¹, Sudeh Izadmehr¹, Josep Domingo-Domenech¹, Carlos Cordon-Cardo^{1 1}Department of Pathology, Icahn School of Medicine at Mount Sinai We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

• Medicine

  
  Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34⁺ Cells Using Valproic Acid Luena Papa¹, Mansour Djedaini¹, Ronald Hoffman^{1 1}Division of Hematology/Oncology, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai Here, we describe the ex vivo expansion of hematopoietic stem cells from CD34⁺ cells derived from umbilical cord blood treated with a combination of a cytokine cocktail and VPA. This method leads to a significant degree of ex vivo expansion of primitive HSCs for either clinical or laboratory applications.

• Medicine

  
  The Left Pneumonectomy Combined with Monocrotaline or Sugen as a Model of Pulmonary Hypertension in Rats Michael G. Katz¹, Anthony S. Fargnoli¹, Sarah M. Gubara¹, Malik Bisserier¹, Yassine Sassi¹, Charles R. Bridges¹, Roger J. Hajjar¹, Lahouaria Hadri^{1 1}Cardiovascular Research Center, Department of Cardiology, Icahn School of Medicine at Mount Sinai The rodent left pneumonectomy is a valuable technique in pulmonary hypertension research. Here, we present a protocol to describe the rat pneumonectomy procedure and postoperative care to ensure minimal morbidity and mortality.

• Bioengineering

  
  A Tripeptide-Stabilized Nanoemulsion of Oleic Acid Sylwia A. Dragulska¹, Marek T. Wlodarczyk^1,2, Mina Poursharifi^1,3, John A. Martignetti^4,5,6, Aneta J. Mieszawska^{1,2,3 1}Department of Chemistry, Brooklyn College, The City University of New York, ²Ph.D. Program in Chemistry, The Graduate Center of The City University of New York, ³Ph.D. Program in Biochemistry, The Graduate Center of The City University of New York, ⁴Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, ⁵Women's Health Research Institute, Icahn School of Medicine at Mount Sinai, ⁶Laboratory for Translational Research, Western Connecticut Health Network This protocol describes an efficient method to synthesize a nanoemulsion of an oleic acids-platinum(II) conjugate stabilized with a lysine-tyrosine-phenylalanine (KYF) tripeptide. The nanoemulsion forms under mild synthetic conditions via self-assembly of the KYF and the conjugate.

• Bioengineering

  
  Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers Anna M. Chiarella¹, Tiffany A. Wang², Kyle V. Butler³, Jian Jin³, Nathaniel A. Hathaway^{4 1}Chemical Biology and Medicinal Chemistry, Center for Integrative Chemical Biology and Drug Discovery, Curriculum in Genetics and Molecular Biology, University of North Carolina, ²College of Arts and Sciences, Chemical Biology and Medicinal Chemistry, Center for Integrative Chemical Biology and Drug Discovery, University of North Carolina, ³Chemical Biology and Drug Discovery, Pharmacological Sciences and Oncological Sciences, Icahn School of Medicine at Mount Sinai, ⁴Chemical Biology and Medicinal Chemistry, Center for Integrative Chemical Biology and Drug Discovery, Curriculum in Genetics and Molecular Biology, Lineberger Comprehensive Cancer Center, University of North Carolina Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

• Immunology and Infection

  
  A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion Anthony M. Esposito^1,2, Alexandra Y. Soare¹, Foramben Patel¹, Namita Satija¹, Benjamin K. Chen¹, Talia H. Swartz^{1 1}Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, Immunology Institute, ²Department of Biology, New Jersey City University We describe a cell-based assay to report on HIV-1 fusion via the expression of green fluorescent protein detectable by flow cytometry or fluorescence microscopy. It can be used to test inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems.

• Immunology and Infection

  
  A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies Mark J. Bailey^1,2, Felix Broecker¹, Paul E. Leon^1,2, Gene S. Tan^{3 1}Department of Microbiology, Icahn School of Medicine at Mount Sinai, ²Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, ³Department of Infectious Disease, J. Craig Venter Institute We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

• Developmental Biology

  
  Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos Evan Bardot^1,2,3, Nikos Tzavaras⁴, Deanna L. Benson^5,6, Nicole C. Dubois^{1,2,3 1}Cell, Developmental, and Regenerative Biology Department, Icahn School of Medicine at Mount Sinai, ²Mindich Child Health and Development Institute, Icahn School of Medicine at Mount Sinai, ³Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, ⁴Microscopy Core, Icahn School of Medicine at Mount Sinai, ⁵Department of Neuroscience, Icahn School of Medicine at Mount Sinai, ⁶Friedman Brain Institute, Icahn School of Medicine at Mount Sinai Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

• Cancer Research

  
  Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel Lisa Mohr¹, Marc Carceles-Cordon¹, Jungreem Woo¹, Carlos Cordon-Cardo¹, Josep Domingo-Domenech¹, Veronica Rodriguez-Bravo^{1,2 1}Department of Pathology, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, ²Department of Oncological Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai Resistance to cancer therapies contributes to disease progression and death. Determining the mechanistic underpinnings of resistance is crucial for improving therapeutic response. This manuscript details the protocol to generate taxane-resistant cell models of prostate cancer (PC) to help dissecting the pathways involved in progression to Docetaxel resistance in PC patients.

• Behavior

  
  Novel Object Recognition Test for the Investigation of Learning and Memory in Mice Lindsay M. Lueptow^{1 1}Pharmacological Sciences, Icahn School of Medicine at Mount Sinai The object recognition test (ORT) is a simple and efficient assay for evaluating learning and memory in mice. The methodology is described below.

• Immunology and Infection

  
  Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies Paul E. Leon^1,2, Teddy John Wohlbold^1,2, Wenqian He^1,2, Mark J. Bailey^1,2, Carole J. Henry³, Patrick C. Wilson³, Florian Krammer¹, Gene S. Tan^{1 1}Department of Microbiology, Icahn School of Medicine at Mount Sinai, ²Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, ³The Department of Medicine, Section of Rheumatology, The Knapp Center for Lupus and Immunology Research, The University of Chicago We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

• Immunology and Infection

  
  Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity Jordi Ochando^1,2, Patricia Conde^{1,2 1}Department of Medicine, Icahn School of Medicine at Mount Sinai, ²Immunología de Trasplantes, Centro Nacional de Microbiología, Instituto de Salud Carlos III Macrophages are plastic cells of the hematopoietic system that have a crucial role in protective immunity and homeostasis. In this report, we describe optimized in vitro techniques to phenotypically and functionally characterize graft-infiltrating regulatory macrophages that accumulate in the transplanted organ under tolerogenic conditions.

• Cancer Research

  
  A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines Jun Tang¹, Carlos Pérez-Medina^1,2, Yiming Zhao², Ahmad Sadique¹, Willem J. M. Mulder², Thomas Reiner^{1 1}Department 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.

• Developmental Biology

  
  Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program Michael G. Daniel^1,2,3, Carlos-Filipe Pereira⁴, Jeffrey M. Bernitz^1,2,3, Ihor R. Lemischka^1,3,5, Kateri Moore^{1,3 1}Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, ²The Graduate School of Biomedical Science, Icahn School of Medicine at Mount Sinai, ³Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, ⁴Center for Neuroscience and Cell Biology, University of Coimbra, ⁵Department of Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount Sinai The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

• Developmental Biology

  
  Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae Theresa A. Reimels^1,2, Cathie M. Pfleger^{1,2 1}The Graduate School of Biomedical Sciences, The Icahn School of Medicine at Mount Sinai, ²Department of Oncological Sciences, The Icahn School of Medicine at Mount Sinai Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells.

• Neuroscience

  
  HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice Terrell Holloway¹, Jose L. Moreno¹, Javier González-Maeso^{1,2,3,4 1}Department of Psychiatry, Icahn School of Medicine at Mount Sinai, ²Department of Neurology, Icahn School of Medicine at Mount Sinai, ³Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, ⁴Department of Physiology and Biophysics, Virginia Commonwealth University Medical School This article describes how to inject viral vectors into the mouse frontal cortex to test behavioral assays that require GPCR heteromeric formation.

• Neuroscience

  
  Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters (CNiFERs) for Optical Detection of Neurotransmitters In Vivo Emre Lacin¹, Arnaud Muller², Marian Fernando¹, David Kleinfeld^2,3, Paul A. Slesinger^{1 1}Department of Neuroscience, Icahn School of Medicine at Mount Sinai, ²Department of Physics, University of California, San Diego, ³Section of Neurobiology, University of California, San Diego We present a protocol to create cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) for the optical detection of volumetric neurotransmitter release.

• Bioengineering

  
  Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy Timothy J. Cashman¹, Rebecca Josowitz², Bruce D. Gelb², Ronald A. Li^1,3, Nicole C. Dubois², Kevin D. Costa^{1 1}Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, ²The Mindich Child Health and Development Institute, Icahn School of Medicine at Mount Sinai, ³Stem Cell & Regenerative Medicine Consortium, LKS Faculty of Medicine, University of Hong Kong This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

• Biology

  
  Identification of Kinesin-1 Cargos Using Fluorescence Microscopy Clement M. Lee^{1 1}Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai Here, a protocol is presented to identify Kinesin-1 cargos. A motorless mutant of the Kinesin-1 heavy chain (KIF5B) aggregates in the cytoplasm and induces aggregation of its cargos. Both aggregates are detected under fluorescence microscopy. A similar strategy can be employed to identify cargos of other motor proteins.

• Medicine

  
  Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells Estrelania S. Williams¹, Veronica Rodriguez-Bravo³, Uma Chippada-Venkata², Janis De Ia Iglesia-Vicente¹, Yixuan Gong², Matthew Galsky², William Oh², Carlos Cordon-Cardo¹, Josep Domingo-Domenech^{1 1}Department of Pathology, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, ²Department of Hematology/Oncology, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, ³Molecular Biology Program, Memorial Sloan-Kettering Cancer Center This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

• Medicine

  
  The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma Michael Li^1,2, Ravi R. Pathak⁵, Esther Lopez-Rivera³, Scott L. Friedman¹, Julio A. Aguirre-Ghiso⁴, Andrew G. Sikora^{5 1}Department of Medicine, Division of Liver Diseases, Icahn School of Medicine at Mount Sinai, ²Department of Otolaryngology, Icahn School of Medicine at Mount Sinai, ³Division of Nephrology, Columbia University College of Physicians and Surgeons, ⁴Departments of Medicine, Hematology and Medical Oncology, Icahn School of Medicine at Mount Sinai, ⁵Bobby R. Alford Department of Otolaryngology - Head and Neck Surgery, Baylor College of Medicine The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

• Medicine

  
  A Multicenter MRI Protocol for the Evaluation and Quantification of Deep Vein Thrombosis Venkatesh Mani¹, Nadia Alie¹, Sarayu Ramachandran¹, Philip M. Robson¹, Cecilia Besa¹, Gregory Piazza², Michele Mercuri³, Michael Grosso³, Bachir Taouli¹, Samuel Z. Goldhaber², Zahi A. Fayad^{1 1}Department of Radiology, Translational and Molecular Imaging Institute, Icahn School of Medicine at Mount Sinai, ² The goal of this study is to use magnetic resonance venography with long-circulating gadolinium-based contrast agent and direct thrombus imaging for quantitative evaluation of DVT volume in a multicenter, clinical trial setting. Inter- and intra-observer variability assessments were conducted, and reproducibility of the protocol was determined.

• Medicine

  
  A Guide to Generating and Using hiPSC Derived NPCs for the Study of Neurological Diseases Aaron Topol¹, Ngoc N. Tran¹, Kristen J. Brennand^{1,2 1}Department of Psychiatry, Icahn School of Medicine at Mount Sinai, ²Department of Neuroscience, Icahn School of Medicine at Mount Sinai This protocol describes how neural progenitor cells can be differentiated from human induced pluripotent stem cells, in order to yield a robust and replicative neural cell population, which may be used to identify the developmental pathways contributing to disease pathogenesis in many neurological disorders.

• Bioengineering

  
  Diagnosis of Neoplasia in Barrett’s Esophagus using Vital-dye Enhanced Fluorescence Imaging Daniel P. Perl¹, Neil Parikh¹, Shannon Chang¹, Paul Peng¹, Nadhi Thekkek³, Michelle H. Lee¹, Alexandros D. Polydorides², Josephine Mitcham¹, Rebecca Richards-Kortum³, Sharmila Anandasabapathy^{1 1}Department of Gastroenterology, Icahn School of Medicine at Mount Sinai, ²Department of Pathology, Icahn School of Medicine at Mount Sinai, ³Department of Bioengineering, Rice University Vital-dye enhanced fluorescence imaging (VFI) is a novel in vivo technique that combines high-resolution epithelial imaging with exogenous topical fluorescent contrast to highlight glandular morphology and delineate neoplasia (high grade dysplasia and cancer) in the distal esophagus.

• Medicine

  
  Isolation of Cancer Stem Cells From Human Prostate Cancer Samples Samuel J. Vidal¹, S. Aidan Quinn¹, Janis de la Iglesia-Vicente¹, Dennis M. Bonal¹, Veronica Rodriguez-Bravo², Adolfo Firpo-Betancourt¹, Carlos Cordon-Cardo¹, Josep Domingo-Domenech^{1 1}Department of Pathology, Icahn School of Medicine at Mount Sinai, ²Molecular Biology Program, Memorial Sloan-Kettering Cancer Center The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

• Immunology and Infection

  
  Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System Irina Margine^1,2, Peter Palese^1,3, Florian Krammer^{1 1}Department of Microbiology, Icahn School of Medicine at Mount Sinai, ²Graduate School of Biological Sciences, Icahn School of Medicine at Mount Sinai, ³Department of Medicine, Icahn School of Medicine at Mount Sinai Here we describe a way to express correctly folded and functional influenza virus surface antigens derived from the novel Chinese H7N9 virus in insect cells. The technique can be adapted to express ectodomains of any viral or cellular surface proteins.