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University of Massachusetts Medical School

26 ARTICLES PUBLISHED IN JoVE

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Biology

A Chromatin Assay for Human Brain Tissue
Anouch Matevossian 1, Schahram Akbarian 1
1Psychiatry, Brudnick Neuropsychiatric Research Institute, University of Massachusetts Medical School

Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.

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Biology

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue
Anouch Matevossian 1, Schahram Akbarian 1
1Psychiatry, Brudnick Neuropsychiatric Research Institute, University of Massachusetts Medical School

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

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Biology

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation
Luke Gabriel 1, Zachary Stevens 1, Haley Melikian 1
1University of Massachusetts Medical School

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

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Biology

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.
Nynke L. van Berkum *1, Erez Lieberman-Aiden *2,3,4,5, Louise Williams *2, Maxim Imakaev 6, Andreas Gnirke 2, Leonid A. Mirny 3,6, Job Dekker 1, Eric S. Lander 2,7,8
1Program in Gene Function and Expression, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 2Broad Institute of Harvard and Massachusetts Institute of Technology, 3Division of Health Sciences and Technology, Massachusetts Institute of Technology, 4Program for Evolutionary Dynamics, Department of Organismic and Evolutionary Biology, Department of Mathematics, Harvard University , 5Department of Applied Mathematics, Harvard University , 6Department of Physics, Massachusetts Institute of Technology, 7Department of Systems Biology, Harvard Medical School, 8Department of Biology, Massachusetts Institute of Technology

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

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JoVE Core

Quantitative Autonomic Testing
Peter Novak 1
1Department of Neurology, University of Massachusetts Medical School

Standardized, comprehensive and fully quantitative testing of autonomic functions is described. The autonomic tests consist of evaluation of all three major autonomic domains including cardiovagal, adrenergic and sudomotor. The severity and distribution of dysautonomia is quantitated using Composite Autonomic Severity Scores.

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Biology

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin
Zhiru Guo 1, Kyle Draheim 1, Stephen Lyle 1
1Department of Cancer Biology, University of Massachusetts Medical School

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

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Biology

Isolation of Drosophila melanogaster Testes
Phillip D. Zamore 1, Shengmei Ma 1
1Department of Biochemistry & Molecular Pharmacology and Howard Hughes Medical Institute, University of Massachusetts Medical School

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

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Biology

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos
Ok Hyun Cho 1, Jaime A. Rivera-Pérez 1, Anthony N. Imbalzano 1
1Department of Cell Biology, University of Massachusetts Medical School

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

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Biology

Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay
Miroslav Koulnis *1, Ramona Pop *1, Ermelinda Porpiglia *1, Jeffrey R. Shearstone *1, Daniel Hidalgo 1, Merav Socolovsky 1
1Department of Pediatrics and Department of Cancer Biology, University of Massachusetts Medical School

A flow-cytometric method for identification and molecular analysis of differentiation-stage-specific murine erythroid progenitors and precursors, directly in freshly –harvested mouse bone marrow, spleen or fetal liver. The assay relies on cell-surface markers CD71, Ter119, and cell size.

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Neuroscience

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster
Motojiro Yoshihara 1
1Department of Neurobiology, University of Massachusetts Medical School

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

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Medicine

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model
Sharanya Iyengar 1, Yariv Houvras 2,3, Craig J. Ceol 1
1Program in Molecular Medicine and Department of Cancer Biology, University of Massachusetts Medical School, 2Departments of Surgery and Medicine, Weill Cornell Medical College , 3Departments of Surgery and Medicine, New York Presbyterian Hospital

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

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Neuroscience

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons
Luke R. Gabriel 1, Sijia Wu 1, Haley E. Melikian 2
1Program in Neuroscience, Graduate School of Biomedical Sciences, University of Massachusetts Medical School, 2Brudnick Neuropsychiatric Research Institute, Department of Psychiatry, University of Massachusetts Medical School

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission. To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons. Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

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Medicine

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells
Sachi Horibata 1, Tommy V. Vo 2, Venkataraman Subramanian 3, Paul R. Thompson 3, Scott A. Coonrod 1
1Department of Baker Institute for Animal Health, Cornell University, 2Department of Molecular Biology and Genetics, Cornell University, 3Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

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Biology

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes
Thomas E. Akie 1, Marcus P. Cooper 1
1Division of Cardiovascular Medicine, Department of Medicine, University of Massachusetts Medical School

De novo lipogenesis and β-fatty acid oxidation constitute key metabolic pathways in hepatocyte, pathways that are perturbed in several metabolic disorders, including fatty liver disease. Here we demonstrate isolation of mouse primary hepatocytes and describe quantification of β-fatty acid oxidation and lipogenesis.

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Immunology and Infection

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood
Pauline Teuffel 1, Miriam Goebel-Stengel 1,2, Tobias Hofmann 1, Philip Prinz 1, Sophie Scharner 1, Jan L. Körner 3, Carsten Grötzinger 3, Matthias Rose 1, Burghard F. Klapp 1, Andreas Stengel 1
1Charité Center for Internal Medicine and Dermatology, Division General Internal and Psychosomatic Medicine, Charité Universitätsmedizin Berlin, Campus Benjamin Franklin, 2Department of Internal Medicine, Institute of Neurogastroenterology and Motility, Martin-Luther Hospital, Academic Teaching Institution of Charité Universitätsmedizin Berlin, 3Department of Hepatology and Gastroenterology, Molecular Cancer Research Center (MKFZ), Campus Virchow-Klinikum, Charité-Universitätsmedizin Berlin

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

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Developmental Biology

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment
Destiny L. Baars 1, Kendra A. Takle *1,2, Jonathon Heier *1,3, Francisco Pelegri 1
1Laboratory of Genetics, University of Wisconsin, 2Department of Neurobiology, University of Massachusetts Medical School, 3Interdisciplinary Biomedical Graduate Program, University of Pittsburgh School of Medicine

A modified protocol for ploidy manipulation uses a heat shock to induce a one-cycle stall in cytokinesis in the early embryo. This protocol is demonstrated in the zebrafish but may be applicable to other species.

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Biochemistry

Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes
Megan E. Dowdle 1, Susanne Blaser Imboden 1, Sookhee Park 1, Sean P. Ryder 2, Michael D. Sheets 1
1Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, 2Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School

Native polyacrylamide gel electrophoresis is a fundamental tool for analyzing RNA-protein interactions. Traditionally most experiments have used vertical gels. However, horizontal gels provide several advantages, such as the opportunity to monitor complexes during electrophoresis. We provide a detailed protocol for generating and using horizontal native gel electrophoresis.

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Biology

Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells
Katilyn Knutson *1, Peter R. Strege *1, Joyce Li 2, Andrew B. Leiter 2, Gianrico Farrugia 1, Arthur Beyder 1
1Enteric Neuroscience Program, Division of Gastroenterology & Hepatology,Department of Physiology & Biomedical Engineering, Mayo Clinic, 2Division of Gastroenterology, Department of Medicine, University of Massachusetts Medical School

Enterochromaffin (EC) cells comprise a small subset of gastrointestinal epithelial cells. EC cells are electrically excitable and release serotonin, yet difficulties in culturing and identifying EC cells have limited physiological studies. The method presented here establishes a primary culture model amenable to examination of single EC cells by electrophysiology.

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Neuroscience

Direct Intrathecal Injection of Recombinant Adeno-associated Viruses in Adult Mice
Dongxiao Li *1, Yundu Li *2, Yunyun Tian 1, Zuoshang Xu 3, Yansu Guo 1,4
1Department of Neurology, Second Hospital of Hebei Medical University, 2No.2 Middle School of Shijiazhuang, 3Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 4Key Laboratory of Hebei Neurology

Here we present a direct intrathecal injection technique using 1% lidocaine hydrochloride in a viral solution to ensure efficient adeno-associated virus delivery to small animals and establish a scoring system to predict transduction efficiency in the central nervous system according to the degree of transient weakness induced by lidocaine.

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Immunology and Infection

Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting
Jean K. Millet 1,2, Tiffany Tang 3, Lakshmi Nathan 3, Javier A. Jaimes 4, Hung-Lun Hsu 3,5, Susan Daniel 3, Gary R. Whittaker 1
1Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, 2INRA, Virologie et Immunologie Moléculaires, 3Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, 4Department of Microbiology, College of Agricultural and Life Sciences, Cornell University, 5Horae Gene Therapy Center, University of Massachusetts Medical School

Here, we present a protocol to generate pseudotyped particles in a BSL-2 setting incorporating the spike protein of the highly pathogenic viruses Middle East respiratory syndrome and severe acute respiratory syndrome coronaviruses. These pseudotyped particles contain a luciferase reporter gene allowing quantification of virus entry into target host cells.

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Biochemistry

Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells
Shellaina J.V. Gordon *1, Yao Xiao *2, Amanda L. Paskavitz 3, Napoleón Navarro-Tito 4, Juan G. Navea 2, Teresita Padilla-Benavides 1
1Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 2Department of Chemistry, Skidmore College, 3Candiac MR Center, Beth Israel Deaconess Medical Center, 4Facultad de Ciencias Químico Biológicas, Universidad Autónoma de Guerrero

Cultured primary or established cell lines are commonly used to address fundamental biological and mechanistic questions as an initial approach before using animal models. This protocol describes how to prepare whole cell extracts and subcellular fractions for studies of zinc (Zn) and other trace elements with atomic absorbance spectroscopy.

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Medicine

Sucrose Preference and Novelty-Induced Hypophagia Tests in Rats using an Automated Food Intake Monitoring System
Martha Anna Schalla *1, Stephanie Gladys Kühne *1, Tiemo Friedrich 1, Vivien Hanel 1, Peter Kobelt 1, Miriam Goebel-Stengel 1,2,3, Matthias Rose 1,4, Andreas Stengel 1,3
1Charité Center for Internal Medicine and Dermatology, Department for Psychosomatic Medicine, Charité-Universitätsmedizin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, 2Department of Internal Medicine, Helios Clinic, 3Department of Psychosomatic Medicine and Psychotherapy, University Hospital Tübingen, 4Department of Quantitative Health Sciences, University of Massachusetts Medical School

Presented here is a protocol to study depression-like and anhedonic behavior in rats. It combines two well-established behavioral methods, the sucrose preference and novelty-induced hypophagia tests, with an automated food and liquid intake monitoring system, to indirectly investigate rodent behavior using surrogate parameters.

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Neuroscience

Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons
David G. Keener *1,2, Amy Cheung *1,3, Kensuke Futai 1
1Brudnick Neuropsychiatric Research Institute, Department of Neurobiology, University of Massachusetts Medical School, 2Interdisciplinary Graduate Program, University of Massachusetts Medical School, 3UMMS MD/PhD Program, University of Massachusetts Medical School

Presented here is a protocol for single-cell electroporation that can deliver genes in both excitatory and inhibitory neurons across a range of in vitro hippocampal slice culture ages. Our approach provides precise and efficient expression of genes in individual cells, which can be used to examine cell-autonomous and intercellular functions.

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Neuroscience

Isolation of Adult Human Astrocyte Populations from Fresh-Frozen Cortex Using Fluorescence-Activated Nuclei Sorting
Zarmeen Mussa 1,2, Jessica Tome-Garcia 1,2, Yan Jiang 3, Schahram Akbarian 2,4, Nadejda M. Tsankova 1,2
1Department of Pathology, Icahn School of Medicine at Mount Sinai, 2Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, 3Institutes of Brian Science, Fudan University, 4Department of Psychiatry, Icahn School of Medicine at Mount Sinai

We have developed a method that enriches for and isolates human astrocyte populations from fresh-frozen tissue for use in downstream molecular analyses.

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Genetics

Capturing Chromosome Conformation Across Length Scales
Liyan Yang 1, Betul Akgol Oksuz 1, Job Dekker 1,2, Johan Harmen Gibcus 1
1Department of Systems Biology, University of Massachusetts Medical School, 2Howard Hughes Medical Institute

Hi-C 3.0 is an improved Hi-C protocol that combines formaldehyde and disuccinimidyl glutarate crosslinkers with a cocktail of DpnII and DdeI restriction enzymes to increase the signal-to-noise ratio and the resolution of chromatin interaction detection.

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Developmental Biology

Identification and Isolation of Burst-Forming Unit and Colony-Forming Unit Erythroid Progenitors from Mouse Tissue by Flow Cytometry
Aishwarya Swaminathan 1, Yung Hwang 1, Ashley Winward 1, Merav Socolovsky 1
1Department of Molecular, Cell and Cancer Biology, UMASS Chan Medical School

Here, we describe a novel flow cytometric method for prospective isolation of early burst-forming unit erythroid (BFU-e) and colony-forming unit erythroid (CFU-e) progenitors directly from fresh mouse bone marrow and spleen. This protocol, developed based on single-cell transcriptomic data, is the first to isolate all the tissue's erythroid progenitors with high purity.

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