Weill Cornell Medical College 30 articles published in JoVE Medicine Point-Of-Care Ultrasound Screening for Proximal Lower Extremity Deep Venous Thrombosis Rebecca G. Theophanous1, Vinca W. Chow2, David L. Convissar3, Stephen C. Haskins4,5, Robert A. Jones6, Hari K. P. Kalagara7, Yuriy S. Bronshteyn8 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. Biology Extraction, Labeling, and Purification of Lineage-Specific Cells from Human Antral Follicles Limor Man1, Nicole Lustgarten Guahmich1, Eleni Kallinos1, Laury Arazi1, Zev Rosenwaks1,2, Daylon James1,2 1Center for Reproductive Medicine and Infertility, Weill Cornell Medical College, 2Tri-Institutional Stem Cell Derivation Laboratory, Weill Cornell Medical College Here, we present protocols for the identification and purification of ovarian cells from antral follicles. We elaborate on methods for processing whole ovaries for the cryopreservation of cortical strips while also harvesting intact antral follicles that are treated enzymatically to liberate multiple follicle resident cell types, including granulosa, theca, endothelial, hematopoietic, and stromal cells. Neuroscience A Psychophysics Paradigm for the Collection and Analysis of Similarity Judgments Suniyya A. Waraich1, Jonathan D. Victor2 1Program in Neuroscience, Weill Cornell Graduate School of Medical Sciences, 2Feil Family Brain and Mind Research Institute, Weill Cornell Medical College The protocol presents an experimental psychophysics paradigm to obtain large quantities of similarity judgments, and an accompanying analysis workflow. The paradigm probes context effects and enables modeling of similarity data in terms of Euclidean spaces of at least five dimensions. Immunology and Infection Performing Colonoscopic-Guided Pinch Biopsies in Mice and Evaluating Subsequent Tissue Changes David C. Montrose1,2, Erin M. McNally3, Erika Sue3, Andrew J. Dannenberg3 1Department of Pathology, Renaissance School of Medicine, Stony Brook University, Stony Brook, NY, 2Stony Brook Cancer Center, Stony Brook, NY, 3Department of Medicine, Weill Cornell Medicine, New York, NY Here, we provide a detailed description of the procedure to induce colonoscopic-guided pinch biopsies in mice and track wound closure in real time. Additionally, methods for the preparation of tissues for histological, immunohistochemical and molecular analyses of the wound bed are provided. Biochemistry Using an Extracellular Flux Analyzer to Measure Changes in Glycolysis and Oxidative Phosphorylation during Mouse Sperm Capacitation Melanie Balbach1, Jochen Buck1, Lonny R. Levin1 1Department of Pharmacology, Weill Cornell Medical College We describe the application of an extracellular flux analyzer to monitor real-time changes in glycolysis and oxidative phosphorylation during mouse sperm capacitation. Engineering Rapid Manufacturing of Thin Soft Pneumatic Actuators and Robots Amir Ali Amiri Moghadam1,2,3, Alexandre Caprio1,2, Seyedhamidreza Alaie1,2, James K. Min1,2, Simon Dunham1,2, Bobak Mosadegh1,2 1Dalio Institute of Cardiovascular Imaging, New York-Presbyterian Hospital, Weill Cornell Medicine, 2Department of Radiology, Weill Cornell Medicine, 3Mechatronics Engineering Department, Kennesaw State University This protocol describes a method for rapid manufacturing of soft pneumatic actuators and robots with a thin form factor. The fabrication method starts with lamination of thermoplastic polyurethane (TPU) sheets followed by laser cutting/welding of a two-dimensional pattern to form actuators and robots. Environment Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples Zuha Anwar*1, Samantha B. Regan*1, Jennifer Linden1 1Brain and Mind Institute, Weill Cornell Medical College The objective of this protocol is to detect different Clostridium perfringens toxinotypes in locally purchased foods, particularly epsilon toxin producing strain types B and D, without the use of anaerobic chambers. Cancer Research Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor Targeting Chrysafis Andreou1, Anton Oseledchyk1, Fay Nicolson1, Naxhije Berisha1,2, Suchetan Pal1, Moritz F. Kircher1,3,4,5,6,7 1Department of Radiology, Memorial Sloan Kettering Cancer Center, 2Department of Chemistry, The Graduate Center of the City University of New York, 3Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, 4Center for Molecular Imaging and Nanotechnology (CMINT), Memorial Sloan Kettering Cancer Center, 5Gerstner Sloan Kettering Graduate School of Biomedical Sciences, 6Department of Radiology, Weill Cornell Medical College of Cornell University, 7Dana-Farber Cancer Institute and Harvard Medical Center Ovarian cancer forms metastases throughout the peritoneal cavity. Here, we present a protocol to make and use folate-receptor targeted surface-enhanced resonance Raman scattering nanoprobes that reveal these lesions with high specificity via ratiometric imaging. The nanoprobes are administered intraperitoneally to living mice, and the derived images correlate well with histology. Chemistry Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates Maria Davydova1, Guillaume Dewaele Le Roi1,2, Pierre Adumeau1, Brian M. Zeglis1,2,3,4 1Department of Chemistry, Hunter College of the City University of New York, 2Ph.D. Program in Chemistry, Graduate Center of the City University of New York, 3Department of Radiology, Memorial Sloan Kettering Cancer Center, 4Department of Radiology, Weill Cornell Medical College In this protocol, we will describe the synthesis of PODS, a phenyoxadiazolyl methyl sulfone-based reagent for the site-selective attachment of cargos to the thiols of biomolecules, particularly antibodies. In addition, we will describe the synthesis and characterization of a PODS-bearing bifunctional chelator and its conjugation to a model antibody. Chemistry Pretargeted Radioimmunotherapy Based on the Inverse Electron Demand Diels-Alder Reaction Rosemery Membreno1,2, Brendon E. Cook1,2,3, Brian M. Zeglis1,2,3,4 1Department of Chemistry, Hunter College of the City University of New York, 2Ph.D. Program in Chemistry, Graduate Center of the City University of New York, 3Department of Radiology, Memorial Sloan Kettering Cancer Center, 4Department of Radiology, Weill Cornell Medical College This protocol describes the synthesis and characterization of a trans-cyclooctene (TCO)-modified antibody and a 177Lu-labeled tetrazine (Tz) radioligand for pretargeted radioimmunotherapy (PRIT). In addition, it details the use of these two constructs for in vivo biodistribution and longitudinal therapy studies in a murine model of colorectal cancer. Bioengineering Co-transplantation of Human Ovarian Tissue with Engineered Endothelial Cells: A Cell-based Strategy Combining Accelerated Perfusion with Direct Paracrine Delivery Limor Man1, Laura Park1, Richard Bodine1, Michael Ginsberg2, Nikica Zaninovic3, Glenn Schattman1, Robert E. Schwartz4, Zev Rosenwaks1,3, Daylon James1,3 1Center for Reproductive Medicine and Infertility, Weill Cornell Medical College, 2Angiocrine Biosciences, Inc., 3Tri-Institutional Stem Cell Derivation Laboratory, Weill Cornell Medical College, 4Joan and Sanford I. Weill Department of Medicine, Weill Cornell Medical College For some patients, the only option for fertility preservation is cryopreservation of ovarian tissue. Unfortunately, delayed revascularization undermines follicular viability. Here, we present a protocol to co-transplant human ovarian tissue with endothelial cells for utilization as a cell-based strategy combining accelerated perfusion with a direct paracrine delivery of bioactive molecules. Cancer Research Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model George Zhang1, Yudan Chi1, Yi-Chieh Nancy Du1 1Department of Pathology and Laboratory Medicine, Weill Cornell Medicine We present a protocol to demonstrate a novel somatic gene transfer system utilizing RIP-Tag; RIP-tva mouse model to study the function of genes in metastasis. The avian retroviruses are delivered intracardiacally to ensure gene transfer into pre-malignant, noninvasive lesions of pancreatic β cells in adult mice. Behavior The Knob Supination Task: A Semi-automated Method for Assessing Forelimb Function in Rats Samuel D. Butensky1, Thelma Bethea1, Joshua Santos1, Anil Sindhurakar1, Eric Meyers2,3, Andrew M. Sloan2,3, Robert L. Rennaker II2,3, Jason B. Carmel1,4,5 1Burke Medical Research Institute, 2Texas Biomedical Center, The University of Texas at Dallas, 3Erik Jonsson School of Engineering and Computer Science, The University of Texas at Dallas, 4Brain and Mind Research Institute, Weill Cornell Medical College, 5Departments of Neurology and Pediatrics, Weill Cornell Medical College This manuscript describes a semi-automated task that quantifies supination in rats. Rats reach, grasp, and supinate a spherical manipulandum. The rat is rewarded with a pellet if the turn angle exceeds a criterion set by the user. This task increases throughput, sensitivity to injury, and objectivity compared to traditional tasks. Genetics Flow-sorting and Exome Sequencing of the Reed-Sternberg Cells of Classical Hodgkin Lymphoma Jonathan B. Reichel1, Jason McCormick2, Jonathan R. Fromm3, Olivier Elemento4, Ethel Cesarman5, Mikhail Roshal6 1Innovation Laboratory, Center for Molecular Oncology, Memorial Sloan Kettering Cancer Center, 2Flow-Sorting Core Facility, Weill Cornell Medical College, 3Department of Laboratory Medicine, University of Washington, 4Department of Physiology and Biophysics, Institute for Computational Biomedicine, Weill Cornell Medical College, 5Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, 6Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center Here, we describe a combined flow cytometric cell sorting and low-input, next-generation library construction protocol designed to produce high-quality, whole-exome data from the Hodgkin Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (CHL). Biochemistry A Tandem Liquid Chromatography–Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus David J. Samuels1, Zhe Wang2, Kyu Y. Rhee2,3, Shaun R. Brinsmade1 1Department of Biology, Georgetown University, 2Division of Infectious Diseases, Weill Cornell Medical College, 3Department of Medicine, Weill Cornell Medical College Here we describe a protocol for the extraction of metabolites from Staphylococcus aureus and their subsequent analysis via liquid chromatography and mass spectrometry. Bioengineering A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting Gabriel E. Büchel1,2, Brandon Carney1,3, Jun Tang1, Brian M. Zeglis1,3, Jörg Eppinger2, Thomas Reiner1,4 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. Immunology and Infection Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria Ben Gold1, Julia Roberts*1, Yan Ling*1, Landys Lopez Quezada1, Jou Glasheen1, Elaine Ballinger1, Selin Somersan-Karakaya2, Thulasi Warrier1, Carl Nathan1 1Departments of Microbiology & Immunology, Weill Cornell Medical College, 2Medicine, Weill Cornell Medical College The charcoal agar resazurin assay (CARA) is a semi-quantitative, medium-throughput method to assess activity of test agents against mycobacteria that are replicating, non-replicating, or both. The CARA permits rapid evaluation of time- and concentration-dependent activity and identifies parameters to pursue by colony forming unit (CFU) assays. Biochemistry A Fluorescence-based Assay of Phospholipid Scramblase Activity Birgit Ploier1, Anant K. Menon1 1Department of Biochemistry, Weill Cornell Medical College We describe a fluorescence-based assay to measure phospholipid scrambling in large unilamellar liposomes reconstituted with opsin. Medicine VDJ-Seq: Deep Sequencing Analysis of Rearranged Immunoglobulin Heavy Chain Gene to Reveal Clonal Evolution Patterns of B Cell Lymphoma Yanwen Jiang1,2, Kui Nie3, David Redmond2, Ari M. Melnick1, Wayne Tam3, Olivier Elemento2 1Department of Medicine, Weill Cornell Medical College, 2Institute for Computational Biomedicine, Weill Cornell Medical College, 3Department of Pathology and Laboratory Medicine, Weill Cornell Medical College This protocol describes an approach to interrogate the recombined immunoglobulin heavy chain VDJ regions of lymphomas by deep-sequencing and retrieve VDJ rearrangement and somatic hypermutation status to delineate clonal architecture of individual tumor. Comparing clonal architecture between paired diagnosis and relapse samples reveals lymphoma relapse clonal evolution modes. Bioengineering Distinctive Capillary Action by Micro-channels in Bone-like Templates can Enhance Recruitment of Cells for Restoration of Large Bony Defect Daniel S. Oh1, Alia Koch1, Sidney Eisig1, Sahng Gyoon Kim2, Yoon Hyuk Kim3, Do-Gyoon Kim4, Jae Hyuck Shim5 1Oral and Maxillofacial Surgery, Columbia University, 2Endodontics, Columbia University, 3Mechanical Engineering, Kyung Hee University, South Korea, 4Orthodontics, The Ohio State University, 5Pathology, Weill Cornell Medical College A step-by-step generic process to create a bone-like template with engineered micro-channels is presented. High absorption and retention capabilities of the template are demonstrated by capillary action via micro-channels. Biology A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters Daniel Basilio1, Alessio Accardi1,2,3 1Department of Anesthesiology, Weill Cornell Medical College, 2Department of Physiology and Biophysics, Weill Cornell Medical College, 3Department of Biochemistry, Weill Cornell Medical College Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described. Biology Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution Francine E. Garrett-Bakelman*1, Caroline K. Sheridan*1, Thadeous J. Kacmarczyk1, Jennifer Ishii1, Doron Betel1,2, Alicia Alonso1, Christopher E. Mason3, Maria E. Figueroa4, Ari M. Melnick1 1Department of Medicine, Weill Cornell Medical College, 2Institute for Computational Biomedicine, Weill Cornell Medical College, 3Department of Physiology and Biophysics, Weill Cornell Medical College, 4Department of Pathology, University of Michigan Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions. Bioengineering Porous Silicon Microparticles for Delivery of siRNA Therapeutics Jianliang Shen*1,2, Xiaoyan Wu*1,3, Yeonju Lee1, Joy Wolfram1,4, Zhizhou Yang1, Zong-Wan Mao2, Mauro Ferrari1,5, Haifa Shen1,6 1Department of Nanomedicine, Houston Methodist Research Institute, 2MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry and Chemical Engineering, Sun Yat-sen University, 3Pediatrics Department of Union Hospital, Huazhong University of Science and Technology, 4CAS Key Laboratory for Biomedical Effects of Nanomaterials & Nanosafety, National Center for Nanoscience & Technology of China, 5Department of Medicine, Weill Cornell Medical College, 6Department of Cell and Developmental Biology, Weill Cornell Medical College Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles. Neuroscience Whole-mount Imaging of Mouse Embryo Sensory Axon Projections Kevin J. O’Donovan1, Catherine O’Keeffe1, Jian Zhong1 1Brain and Mind Research Institute, Burke Medical Research Institute, Weill Medical College of Cornell University We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice. Medicine Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics Adriana D. Corben*1, Mohammad M. Uddin*2, Brooke Crawford3, Mohammad Farooq4, Shanu Modi5, John Gerecitano5, Gabriela Chiosis2, Mary L. Alpaugh6 1Department of Pathology, Memorial Sloan Kettering Cancer Center, 2Molecular Pharmacology and Chemistry Program, Memorial Sloan Kettering Cancer Center, 3Department of Radiology, Weill Cornell Medical College, 4Department of Medicine, Memorial Sloan Kettering Cancer Center, 5Department of Oncology, Memorial Sloan Kettering Cancer Center, 6Department of Surgery, Memorial Sloan Kettering Cancer Center Established cancer cell lines and xenografts have been the mainstay of cancer research for the past several decades. However, recent evidence suggests that therapeutic response is greatly influenced by the tumor cell microenvironment. Therefore, we have developed an ex vivo analysis of primary tumor specimens for drug development purposes. Biology Protein Isolation from the Developing Embryonic Mouse Heart Valve Region Laura A. Dyer1, Yaxu Wu1, Cam Patterson2 1McAllister Heart Institute, University of North Carolina at Chapel Hill, 2New York-Presbyterian Hospital/Weill-Cornell Medical Center The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis. Neuroscience Subtype-selective Electroporation of Cortical Interneurons Natalia V. De Marco Garcia1,2, Gord Fishell1 1NYU Neuroscience Institute, New York University School of Medicine, 2Brain and Mind Research Institute, Weill Cornell Medical College This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex. Medicine In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells Gunjan Gakhar1, Neil H. Bander2, David M. Nanus1 1Department of Medicine, Weill Cornell Medical College, 2Department of Urology, Weill Cornell Medical College Our report describes a unique method to visualize and analyze CTC/EC interactions in prostate cancer under physiological flow conditions. Biology Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry Elena Ivanova1, Abduqodir H Toychiev1, Christopher W Yee1, Botir T Sagdullaev1 1Ophthalmology, Weill Medical College of Cornell University The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components. Behavior Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference Kathryn C. Schierberl1,2, Anjali M. Rajadhyaksha1,2 1Graduate Program in Neuroscience, Weill Cornell Graduate School of Biomedical Sciences, 2Division of Pediatric Neurology, Department of Pediatrics, Weill Cornell Medical College This article describes how to microinject viral vectors into mouse brain and then test in a conditioned place preference paradigm that includes an acquisition, extinction and reinstatement phase.