Albert Einstein College of Medicine 23 articles published in JoVE Immunology and Infection Generation of Monocyte-Derived Dendritic Cells with Differing Sialylated Phenotypes Vanessa C. C. Luz1,2, Zélia Silva1,2, Patrícia Sobral1,2,3, Ankit Tanwar4,5, Rachel L. Paterson6, Paula A. Videira1,2,7 1Associate Laboratory i4HB - Institute for Health and Bioeconomy, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2UCIBIO - Applied Molecular Biosciences Unit, Department of Life Sciences, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 3LAQV and REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, 4Department of Microbiology and Immunology, Albert Einstein College of Medicine, 5Department of Oncology, Albert Einstein College of Medicine, 6Stemmatters, Biotecnologia e Medicina Regenerativa SA, Parque de Ciência e Tecnologia Avepark, Zona Industrial da Gandra, 7CDG & Allies - Professionals and Patient Associations International Network (CDG & Allies - PPAIN), Department of Life Sciences, NOVA School of Science and Technology, Universidade NOVA de Lisboa A unique, comprehensive protocol to generate de-sialylated human monocyte-derived dendritic cells (mo-DCs) from isolated peripheral blood mononuclear cells (PBMCs) using a sialidase treatment is presented. Further, methods to assess the phenotypic and functional characterization of mo-DCs and evaluate how sialidase treatment improves the maturation level of mo-DCs are described. Biochemistry Semi-Automated Phenotypic Analysis of Functional 3D Spheroid Cell Cultures Stephanie Stransky1, Dejauwne Young1, Karoline Mikkelsen2,3, Annemette Præstegaard Thulesen2, Helle Sedighi Frandsen3, Simone Sidoli1 1Department of Biochemistry, Albert Einstein College of Medicine, 2Department of Biochemistry and Molecular Biology, University of Southern Denmark, 3CelVivo ApS We present a protocol for growing high-reproducible spheroids and their phenotypic characterization using image capture and proteomics. Biology Global Level Quantification of Histone Post-Translational Modifications in a 3D Cell Culture Model of Hepatic Tissue Jazmine-Saskya N. Joseph-Chowdhury1, Stephanie Stransky1, Sarah Graff1, Ronald Cutler1, Dejauwne Young1, Julie S. Kim1, Carlos Madrid-Aliste1, Jennifer T. Aguilan1, Edward Nieves1, Yan Sun1, Edwin J. Yoo1, Simone Sidoli1 1Department of Biochemistry, Albert Einstein College of Medicine This protocol outlines how a three-dimensional cell culture system can be used to model, treat, and analyze chromatin modifications in a near-physiological state. Medicine Surgical Techniques to Optimize Ovarian Reserve during Laparoscopic Cystectomy for Ovarian Endometrioma Kathryn Saturnino1,2,3, Osaro Obanor1,2,3, Cynthia Arvizo4, Julian A. Gingold2,3,5 1Department of Obstetrics & Gynecology and Women's Health, Montefiore Medical Center, 2Albert Einstein College of Medicine, 3Department of OB/GYN, 1400 Pelham Pkwy S, Jacobi Medical Center, 4Division of Minimally Invasive Gynecologic Surgery, Department of OB/GYN, Jacobi Medical Center, 5Division of Reproductive Endocrinology and Infertility, Department of Obstetrics & Gynecology and Women's Health, Montefiore Medical Center This protocol presents techniques to laparoscopically excise ovarian endometrioma, to perform adhesiolysis with sparing electrosurgical application, and to employ intraoperative chromopertubation to assess for genital tract patency. This systematic approach will facilitate optimal endometriosis management, guide concomitant adnexal surgeries, and enhance post-surgical fertility outcomes. Developmental Biology A 3-D Tail Explant Culture to Study Vertebrate Segmentation in Zebrafish M. Fethullah Simsek1,3, Ertugrul M. Özbudak1,2,3 1 Here, we present the protocol for 3-D tissue culture of the zebrafish posterior body axis, enabling live study of vertebrate segmentation. This explant model provides control over axis elongation, alteration of morphogen sources, and subcellular resolution tissue-level live imaging. Cancer Research Analysis of Liver Microenvironment During Early Progression of Non-Alcoholic Fatty Liver Disease-Associated Hepatocellular Carcinoma in Zebrafish Cassia Michael1,2, Francisco Juan Martínez-Navarro1,2, Sofia de Oliveira1,2,3,4 1Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, 2Department of Medicine (Hepatology), Albert Einstein College of Medicine, 3Einstein-Mount Sinai Diabetes Research Center, Albert Einstein College of Medicine, 4Marion Bessin Liver Research Center, Albert Einstein College of Medicine Here, we present how to generate a non-alcoholic fatty liver disease (NAFLD)-associated Hepatocellular Carcinoma (HCC) zebrafish model to study the impact of cholesterol surplus on liver microenvironment and immune cell landscape. Developmental Biology Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity Yvon Woappi1,2, Geraldine Ezeka3, Justin Vercellino4, Sean M. Bloos5, Kim E. Creek6, Lucia Pirisi1 1Department of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, 2 Here we describe a protocol for the systematic cultivation of epidermal spheroids in 3D suspension culture. This protocol has wide-ranging applications for use in a variety of epithelial tissue types and for the modeling of several human diseases and conditions. Medicine Real-Time Monitoring of Neurocritical Patients with Diffuse Optical Spectroscopies Rodrigo Menezes Forti1,2, Marilise Katsurayama2,3, Giovani Grisotti Martins1, Lenise Valler2,3, Andrés Quiroga1,2, Luiz Simioni1, Julien Menko4, Antonio L. E. Falcão3, Li Min Li2,5, Rickson C. Mesquita1,2 1Institute of Physics, University of Campinas, 2Brazilian Institute of Neuroscience and Neurotechnology, 3Clinical Hospital, University of Campinas, 4Department of Emergency Medicine, Albert Einstein College of Medicine, 5School of Medical Sciences, University of Campinas Presented here is a protocol for non-invasively monitoring cerebral hemodynamics of neurocritical patients in real-time and at the bedside using diffuse optics. Specifically, the proposed protocol uses a hybrid diffuse optical systems to detect and display real-time information on cerebral oxygenation, cerebral blood flow and cerebral metabolism. Genetics Isolation of Adipose Tissue Nuclei for Single-Cell Genomic Applications Gabrielle J. Benitez1, Kosaku Shinoda1,2,3 1Departments of Medicine, Albert Einstein College of Medicine, 2Departments of Molecular Pharmacology, Albert Einstein College of Medicine, 3Fleischer Institute of Diabetes and Metabolism This publication describes a protocol for the isolation of nuclei from mature adipocytes, purification by fluorescence-activated sorting, and single-cell level transcriptomics. Medicine Updated Technique for Reliable, Easy, and Tolerated Transcranial Electrical Stimulation Including Transcranial Direct Current Stimulation Helen Borges1, Alexandra Dufau1,2, Bhaskar Paneri1, Adam J. Woods3, Helena Knotkova4,5, Marom Bikson1 1Department of Biomedical Engineering, The City College of New York, CUNY, 2Department of Clinical and Health Psychology, Center for Cognitive Aging and Memory, 3McKnight Brain Institute, University of Florida, 4MJHS Institute for Innovation in Palliative Care, 5Department of Family and Social Medicine, Albert Einstein College of Medicine When administering transcranial direct current stimulation (tDCS), reproducible electrode preparation and placement are vital for a tolerated and effective session. The purpose of this article is to demonstrate updated modern setup procedures for the administration of tDCS and related transcranial electrical stimulation techniques, such as transcranial alternating current stimulation (tACS). Neuroscience Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice Ryosuke Fujiki1,2,3,4,9, Joun Y. Lee1,2,10, Julie A. Jurgens1,2,3,7, Mary C. Whitman2,5,6, Elizabeth C. Engle1,2,3,4,5,6,7,8 1 This work presents a protocol to yield homogeneous cell cultures of primary oculomotor, trochlear, and spinal motor neurons. These cultures can be used for comparative analyses of the morphological, cellular, molecular, and electrophysiological characteristics of ocular and spinal motor neurons. Biology Improving the Accuracy of Flow Cytometric Assessment of Mitochondrial Membrane Potential in Hematopoietic Stem and Progenitor Cells Through the Inhibition of Efflux Pumps Claudia Morganti1,2,3, Massimo Bonora1,2,3, Keisuke Ito1,2,3,4 1Ruth L. and David S. Gottesman Institute for Stem Cell and Regenerative Medicine Research, Albert Einstein College of Medicine, 2Departments of Cell Biology and Stem Cell Institute, Albert Einstein College of Medicine, 3Department of Medicine, Albert Einstein College of Medicine, 4Albert Einstein Cancer Center and Diabetes Research Center, Albert Einstein College of Medicine Xenobiotic efflux pumps are highly active in hematopoietic stem and progenitor cells (HSPCs) and cause extrusion of TMRM, a mitochondrial membrane potential fluorescent dye. Here, we present a protocol to accurately measure mitochondrial membrane potential in HSPCs by TMRM in the presence of Verapamil, an efflux pump inhibitor. Cancer Research Assessing Tumor Microenvironment of Metastasis Doorway-Mediated Vascular Permeability Associated with Cancer Cell Dissemination using Intravital Imaging and Fixed Tissue Analysis George S. Karagiannis1,2,3, Jessica M. Pastoriza1,2,4, Lucia Borriello1,2, Rojin Jafari1,2, Anouchka Coste1,2,4, John S. Condeelis1,2,3,4, Maja H. Oktay1,2,3,5, David Entenberg1,2,3 1Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 2Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, 3Integrated Imaging Program, Albert Einstein College of Medicine, 4Department of Surgery, Montefiore Medical Center, 5Department of Pathology, Montefiore Medical Center We describe two methods for assessing transient vascular permeability associated with tumor microenvironment of metastasis (TMEM) doorway function and cancer cell intravasation using intravenous injection of high-molecular weight (155 kDa) dextran in mice. The methods include intravital imaging in live animals and fixed tissue analysis using immunofluorescence. Behavior Using the Race Model Inequality to Quantify Behavioral Multisensory Integration Effects Jeannette R. Mahoney1, Joe Verghese1,2 1Department of Neurology, Division of Cognitive & Motor Aging, Albert Einstein College of Medicine, 2Department of Medicine, Division of Geriatrics, Albert Einstein College of Medicine The current study aims to provide a step-by-step tutorial for calculating the magnitude of multisensory integration effects in an effort to facilitate the production of translational research studies across diverse clinical populations. Genetics Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens Olivier Loudig1,2,3, Christina Liu1,2, Thomas Rohan3, Iddo Z. Ben-Dov4 1Department of Research, Hackensack University Medical Center, 2Department of Medical Sciences, Seton Hall University, 3Department of Epidemiology and Population Health, Albert Einstein College of Medicine, 4Department of Nephrology and Hypertension, Hadassah - Hebrew University Medical Center Formalin-fixed paraffin-embedded specimens represent a valuable source of molecular biomarkers of human diseases. Here we present a laboratory-based cDNA library preparation protocol, initially designed with fresh frozen RNA, and optimized for the analysis of archived microRNAs from tissues stored up to 35 years. Biology Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds Calliandra Maria de-Souza-Silva1, Fernanda Guilhelmelli1, Daniel Zamith-Miranda2,3, Marco Antônio de Oliveira1, Joshua Daniel Nosanchuk2,3, Ildinete Silva-Pereira*1, Patrícia Albuquerque*1,4 1Laboratory of Molecular Biology, Department of Cellular Biology, Institute of Biological Sciences, University of Brasília, 2Department of Microbiology and Immunology, Albert Einstein College of Medicine, 3Division of Infectious Diseases, Department of Medicine, Albert Einstein College of Medicine, 4Faculty of Ceilândia, University of Brasília An easy and adaptable broth microdilution method for screening antifungal compounds and extracts. Bioengineering Synthesis of Functionalized 10-nm Polymer-coated Gold Particles for Endothelium Targeting and Drug Delivery Ming J. Cheng1, Priya Prabakaran1, Rajiv Kumar2,3, Srinivas Sridhar1,2,3, Eno E. Ebong1,4,5 1Department of Chemical Engineering, Northeastern University, 2Nanomedicine Science and Technology Center, Northeastern University, 3Department of Physics, Northeastern University, 4Departments of Bioengineering, Northeastern University, 5Department of Neuroscience, Albert Einstein College of Medicine We describe a method of synthesizing biocompatible 10-nm gold nanoparticles, functionalized by coating poly-ethylene glycol onto the surface. These particles can be used in vitro and in vivo for delivering therapeutics to nanoscale cellular and extracellular spaces that are difficult to access with conventional nanoparticle sizes. Cancer Research Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion Salvatore Coniglio1, Ian Miller2, Marc Symons3, Jeffrey E. Segall4 1New Jersey Center for Science, Technology and Mathematics, Kean University, 2Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, 3The Feinstein Institute for Medical Research at North Shore-LIJ, 4Department of Anatomy and Structural Biology, Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed. Cancer Research Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window Carolina Rodriguez-Tirado1, Takanori Kitamura5, Yu Kato1,2, Jeffery W. Pollard1,2,5, John S. Condeelis3,4, David Entenberg3,4 1Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, 2Department of Obstetrics/Gynecology and Woman’s Health, Albert Einstein College of Medicine, 3Department of Anatomy & Structural Biology, Albert Einstein College of Medicine, 4Gruss-Lipper Biophotonics Center Integrated Imaging Program, Albert Einstein College of Medicine, 5Medical Research Council Centre for Reproductive Health, Queen’s Medical Research Institute, University of Edinburgh This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window. Medicine Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment Allison S. Harney1,2,3,4, Yarong Wang1,3, John S. Condeelis1,3,4, David Entenberg1,3,4 1Department of Anatomy & Structural Biology, Albert Einstein College of Medicine, 2Department of Radiology, Albert Einstein College of Medicine, 3Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, 4Integrated Imaging Program, Albert Einstein College of Medicine This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution. Immunology and Infection An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets Pooja Arora1, Steven A. Porcelli1,2 1Department of Microbiology and Immunology, Albert Einstein College of Medicine, 2Department of Medicine (Rheumatology), Albert Einstein College of Medicine Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells. Biology Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures Tuba Bas1, Leonard H. Augenlicht1,2 1Department of Medicine, Albert Einstein College of Medicine, 2Department of Cell Biology, Albert Einstein College of Medicine Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source. Immunology and Infection Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy Sabriya Stukes1, Arturo Casadevall1 1Department of Microbiology and Immunology, Albert Einstein College of Medicine We describe how to visualize macrophage-C. neoformans (Cn) interactions in real time, with specific emphasis on the process of non-lytic exocytosis using digital light microscopy. Using this technique individually infected macrophages can be studied to ascertain various aspects of this phenomenon.
