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Stony Brook University

29 ARTICLES PUBLISHED IN JoVE

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Neuroscience

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues
Ryan W. O'Meara 1,2, Scott D. Ryan 1, Holly Colognato 3, Rashmi Kothary 1,2,4
1Regenerative Medicine Program, Ottawa Hospital Research Institute, 2Department of Cellular and Molecular Medicine, University of Ottawa , 3Department of Pharmacological Sciences, Stony Brook University, 4Department of Medicine, University of Ottawa

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

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Biology

Measuring Fast Calcium Fluxes in Cardiomyocytes
Urszula Golebiewska 1, Suzanne Scarlata 2
1Department of Biological Sciences and Geology, Queensborough Community College, 2Department of Physiology & Biophysics, Stony Brook University

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

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Immunology and Infection

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry
Frederick A. Rubino 1, Yoon Hyeun Oum 1, Lakshmi Rajaram 1, Yanjie Chu 1, Isaac S. Carrico 1
1Department of Chemistry, Stony Brook University

Adenovirus particles are engineered to contain either the unnatural amino acid analogue azidohomoalanine or the azido sugar O-GlcNAz. The azide group of each is chemoselectively ligated via "click" chemistry reactions as a means of viral surface modification.

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Biology

A Novel Bayesian Change-point Algorithm for Genome-wide Analysis of Diverse ChIPseq Data Types
Haipeng Xing 1, Willey Liao 1,2, Yifan Mo 1,2, Michael Q. Zhang 2,3
1Department of Applied Mathematics & Statistics, Stony Brook University, 2Computational Biology and Bioinformatics, Cold Spring Harbor Laboratory, 3Department of Molecular and Cell Biology, University of Texas at Dallas

Our Bayesian Change Point (BCP) algorithm builds on state-of-the-art advances in modeling change-points via Hidden Markov Models and applies them to chromatin immunoprecipitation sequencing (ChIPseq) data analysis. BCP performs well in both broad and punctate data types, but excels in accurately identifying robust, reproducible islands of diffuse histone enrichment.

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Immunology and Infection

Culturing Microglia from the Neonatal and Adult Central Nervous System
Robert Bronstein *1,2, Luisa Torres *2,3, Jillian C. Nissen *2,3, Stella E. Tsirka 2
1Program in Neuroscience, Stony Brook University, 2Department of Pharmacological Sciences, Stony Brook University, 3Program in Molecular and Cellular Pharmacology, Stony Brook University

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

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Immunology and Infection

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans
Arielle M. Bryan *1, Amir M. Farnoud *1, Visesato Mor 1, Maurizio Del Poeta 1
1Department of Molecular Genetics and Microbiology, Stony Brook University

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

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JoVE Core

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons
Chongbo Zhong 1, David A. Talmage 2, Lorna W. Role 1
1Department of Neurobiology and Behavior, Stony Brook University, 2Department of Pharmacological Science, Stony Brook University

We developed a gene-chimeric preparation of ventral hippocampal – accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

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Biology

A Strategy to Validate the Role of Callose-mediated Plasmodesmal Gating in the Tropic Response
Ritesh Kumar *1, Shu Wei Wu *1, Arya Bagus Boedi Iswanto 1, Dhinesh Kumar 1, Xiao Han 2, Jae-Yean Kim 1
1Division of Applied Life Science (BK21 plus program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, 2Biotechnology Research Institute, Chinese Academy of Agricultural Sciences

This article describes the methods for screening the genes controlling plasmodesmal permeability and hence auxin gradient during tropic response. This includes the measurement of the degree of tropic response in hypocotyl of Arabidopsis thaliana and checking plasmodesmal permeability by 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) loading and finally callose level assessment.

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Biology

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging
Tyler M. Bauman 1,2, Emily A. Ricke 2, Sally A. Drew 3, Wei Huang 3,4, William A. Ricke 2,4
1Division of Urologic Surgery, Washington University in St. Louis School of Medicine, 2Department of Urology, University of Wisconsin School of Medicine and Public Health, 3Department of Pathology and Laboratory Medicine, University of Wisconsin School of Medicine and Public Health, 4O’Brien Urology Research Center, University of Wisconsin School of Medicine and Public Health

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

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Developmental Biology

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis
Patricia Bossert 1, Gerald H. Thomsen 1
1Department of Biochemistry and Cell Biology, Center for Developmental Genetics, Stony Brook University

Here we demonstrate how to induce and monitor regeneration in the Starlet Sea Anemone Nematostella vectensis, a model cnidarian anthozoan. We demonstrate how to amputate and categorize regeneration using a morphological staging system, and we use this system to reveal a requirement for autophagy in regenerating polyp structures.

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Biochemistry

Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay
Jonathan Fischer 1, Roger A. Johnson 2, Elizabeth Boon 1
1Department of Chemistry, Stony Brook University, 2Department of Physiology and Biophysics, Stony Brook University

We report and demonstrate an optimized nitrocellulose binding assay that can be used to quantify autophosphorylation of purified bacterial histidine kinases. Our method has several advantages over traditional SDS-PAGE based techniques, providing a valuable alternative for characterizing these important proteins.

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Behavior

Using a Split-belt Treadmill to Evaluate Generalization of Human Locomotor Adaptation
Erin V.L. Vasudevan 1,2, Rami J. Hamzey 1,2, Eileen M. Kirk 2
1Physical Therapy, School of Health Technology and Management, Stony Brook University, 2Motor Learning Lab, Moss Rehabilitation Research Institute, Einstein Healthcare Network

We describe a protocol for investigating human locomotor adaptation using the split-belt treadmill, which has two belts that can drive each leg at a different speed. We specifically focus on a paradigm designed to test the generalization of adapted locomotor patterns to different walking contexts (e.g., gait speeds, walking environments).

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Biology

Benefits of Cardiac Resynchronization Therapy in an Asynchronous Heart Failure Model Induced by Left Bundle Branch Ablation and Rapid Pacing
Jingfeng Wang *1, Zhenning Nie *1, Haiyan Chen *2, Xianhong Shu 2, Zhaohua Yang 3, Ruiming Yao 1, Yangang Su 1, Junbo Ge 1
1Department of Cardiology, Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, 2Department of Echocardiography, Shanghai Institute of Medical imaging, Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, 3Department of Cardiac surgery, Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University

The establishment of a chronic asynchronous heart failure (HF) model by rapid pacing combined with left bundle branch ablation is presented. Two-dimensional speckle tracking imaging and aortic velocity time integral are applied to validate this stable HF model with left ventricular asynchrony and the benefits of cardiac resynchronization therapy.

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Immunology and Infection

Isolating Lymphocytes from the Mouse Small Intestinal Immune System
Zhijuan Qiu 1, Brian S. Sheridan 1
1Center for Infectious Diseases, Department of Molecular Genetics and Microbiology, Stony Brook University

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer's patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

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JoVE Core

Stress Distribution During Cold Compression of Rocks and Mineral Aggregates Using Synchrotron-based X-Ray Diffraction
Cecilia S.N. Cheung 1,2, Donald J. Weidner 1, Li Li 1, Philip G. Meredith 3, Haiyan Chen 1, Matthew Whitaker 1, Xianyin Chen 4
1Mineral Physics Institute, Department of Geoscience, Stony Brook University, 2Geological Engineering, Department of Civil and Environmental Engineering, University of Wisconsin-Madison, 3Rock and Ice Physics Laboratory, Department of Earth Sciences, University College London, 4Department of Chemistry, Stony Brook University

We report detailed procedures for compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus coupled with synchrotron X-radiation. Such experiments allow quantification of the stress distribution within samples, that ultimately sheds light on compaction processes in geomaterials.

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Cancer Research

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis
Ravan Moret 1, Linh Hellmers 1, Xin Zhang 1, Jessie Gills 2, Nathan Hite 3, Aaron Klinger 3, Grace A. Maresh 1, Daniel Canter 2,4, Stephen Bardot 2,4, David A Margolin 3,4, Li Li 1,4
1Laboratory of Translational Cancer Research, Institute for Translational Research, Ochsner Clinic Foundation, 2Department of Urology, Ochsner Clinic Foundation, 3Department of Colon and Rectal Surgery, Ochsner Clinic Foundation, 4Ochsner Clinical School, University of Queensland, School of Medicine

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

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Biology

Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture
Laurel R. Yohe *1,2, Paolo Devanna *3, Kalina T.J. Davies 4, Joshua H.T. Potter 4, Stephen J. Rossiter 4, Emma C. Teeling 5, Sonja C. Vernes *3,6, Liliana M. Dávalos *2,7
1Department of Geology & Geophysics, Yale University, 2Department of Ecology & Evolution, Stony Brook University, 3Neurogenetics of Vocal Communication, Max Planck Institute for Psycholinguistics, 4School of Biological and Chemical Sciences, Queen Mary University of London, 5School of Biology & Environmental Science, University College Dublin, 6Donders Institute for Brain, Cognition and Behavior, 7Consortium for Inter-Disciplinary Environmental Research, Stony Brook University

This is a protocol for the optimal tissue preparation for genomic, transcriptomic, and proteomic analyses of bats caught in the wild. It includes protocols for bat capture and dissection, tissue preservation, and cell culturing of bat tissue.

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Behavior

3D Kinematic Gait Analysis for Preclinical Studies in Rodents
Jeffrey Wong 1, Prithvi K. Shah 1,2
1Department of Neurobiology and Behavior, Stony Brook University, 2Department of Physical Therapy, Stony Brook University

Presented here is a protocol to collect and analyze three-dimensional kinematics of quadrupedal locomotion in rodents for preclinical studies.

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Medicine

A Rabbit Model of Aqueous-Deficient Dry Eye Disease Induced by Concanavalin A Injection into the Lacrimal Glands: Application to Drug Efficacy Studies
Robert A. Honkanen 1, Liqun Huang 2,3, Basil Rigas 2
1Department of Ophthalmology, Stony Brook University, 2Department of Preventive Medicine, Stony Brook University, 3Medicon Pharmaceuticals, Inc.

This article describes the development of a method to induce acute or chronic dry eye disease in rabbits by injecting concanavalin A to all portions of the orbital lacrimal gland system. This method, superior to those already reported, generates a reproducible, stable model of dry eye suitable for the study of pharmacological agents.

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Engineering

Sandy Soil Improvement through Microbially Induced Calcite Precipitation (MICP) by Immersion
Shihui Liu 1, Kang Du 1, Kejun Wen 1, Wei Huang 2, Farshad Amini 1, Lin Li 3
1Department of Civil & Environmental Engineering, Jackson State University, 2School of Civil Engineering and Architecture, Chongqing University of Science and Technology, 3Department of Civil & Architectural Engineering, Tennessee State University

Here, microbially induced calcite precipitation (MICP) technology is presented to improve soil properties by immersion.

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Medicine

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits
Robert A. Honkanen 1, Liqun Huang 2,3, Wei Huang 1, Basil Rigas 2,4
1Department of Ophthalmology, Stony Brook University, 2Department of Medicine, Stony Brook University, 3Medicon Pharmaceuticals, Inc, 4Department of Preventive Medicine, Stony Brook University

A novel approach is presented to induce chronic dry eye disease in rabbits by surgically removing all orbital lacrimal glands. This method, distinct from those previously reported, produces a stable, reproducible model of aqueous deficient dry eye well suited to study tear physiology and pathophysiology and ocular therapeutics.

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Biochemistry

Induction of Eryptosis in Red Blood Cells Using a Calcium Ionophore
Parnian Bigdelou 1, Amir M. Farnoud 1,2
1Biomedical Engineering Program, Ohio University, 2Department of Chemical and Biomolecular Engineering, Ohio University

A protocol for the induction of eryptosis, programmed cell death in erythrocytes, using the calcium ionophore, ionomycin, is provided. Successful eryptosis is evaluated by monitoring the localization phosphatidylserine in the membrane outer leaflet. Factors affecting the success of the protocol have been examined and optimal conditions provided.

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Medicine

A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo
Yihai Liu 1, Wenfeng Zhang 1, Tingting Hu 1, Jie Ni 1, Biao Xu 1, Wei Huang 1
1Department of Cardiology, Nanjing Drum Tower Hospital, Clinical College of Nanjing Medical University

Described is a protocol to establish a Doxorubicin-induced dilated cardiomyopathy (DCM) model in mice via long-term intraperitoneal injection of Doxorubicin.

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Developmental Biology

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans
Li Li 1, Anna Y. Zinovyeva 1
1Division of Biology, Kansas State University

This method describes a protocol for high-throughput protein extract preparation from Caenorhabditis elegans samples and subsequent co-immunoprecipitation.

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Bioengineering

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells
Michael Tyler Guinn 1,2,3, Damiano Coraci *2, Lesia Guinn *2, Gábor Balázsi 1,2
1Biomedical Engineering Department, Stony Brook University, 2Laufer Center for Physical and Quantitative Biology, Stony Brook University, 3Stony Brook Medical Scientist Training Program

Reliably controlling light-responsive mammalian cells requires the standardization of optogenetic methods. Toward this goal, this study outlines a pipeline of gene circuit construction, cell engineering, optogenetic equipment operation, and verification assays to standardize the study of light-induced gene expression using a negative-feedback optogenetic gene circuit as a case study.

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Medicine

Real-Time Dynamic Navigation System for the Precise Quad-Zygomatic Implant Placement in a Patient with a Severely Atrophic Maxilla
Yihan Shen *1, Qinggang Dai *1, Baoxin Tao *1, Wei Huang 2, Feng Wang 2, Kengliang Lan 1, Yuanyuan Sun 1, Xiaowan Ling 1, Lijun Yan 1, Yueping Wang 1, Yiqun Wu 1
1The 2nd Dental Center, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, College of Stomatology, Shanghai Jiao Tong University, National Center for Stomatology, National Clinical Research Center for Oral Diseases, Shanghai Key Laboratory of Stomatology, 2Department of Dental Implantology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, College of Stomatology, Shanghai Jiao Tong University, National Center for Stomatology, National Clinical Research Center for Oral Diseases, Shanghai Key Laboratory of Stomatology

Here, we present a protocol to achieve precise quad-zygomatic implant placement in patients with severely atrophic maxilla using a real-time dynamic navigation system.

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Cancer Research

Longitudinal Intravital Imaging Through Clear Silicone Windows
Laura Maiorino *1,2,3, Margaret Shevik *1,4,5, José M. Adrover 1, Xiao Han 1,6, Elias Georgas 7,8, John Erby Wilkinson 9, Harrison Seidner 1, Leonie Foerschner 1, David A. Tuveson 1, Yi-Xian Qin 8, Mikala Egeblad 1
1Cold Spring Harbor Laboratory, 2Cold Spring Harbor Laboratory School of Biological Sciences, 3Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 4Medical Scientist Training Program, School of Medicine, Stony Brook University, 5Graduate Program in Pharmacology, Stony Brook University, 6Graduate Program in Genetics, Stony Brook University, 7Graduate Program in Biomedical Engineering, Stony Brook University, 8Department of Biomedical Engineering, Stony Brook University, 9Department of Pathology, University of Michigan

An approach is here presented for long-term intravital imaging using optically clear, silicone windows that can be glued directly to the tissue/organ of interest and the skin. These windows are cheaper and more versatile than others currently used in the field, and the surgical insertion causes limited inflammation and distress to the animals.

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Cancer Research

Establishment and Culture of Patient-Derived Breast Organoids
Disha Aggarwal 1,2, Suzanne Russo 1, Payal Naik 1, Sonam Bhatia 1, David L. Spector 1,2
1Cold Spring Harbor Laboratory, Cold Spring Harbor, 2Genetics Graduate Program, Stony Brook University

A detailed protocol is provided here for establishing human breast organoids from patient-derived breast tumor resections or normal breast tissue. The protocol provides comprehensive step-by-step instructions for culturing, freezing, and thawing human patient-derived breast organoids.

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Neuroscience

Functional Magnetic Resonance Imaging (fMRI) of the Visual Cortex with Wide-View Retinotopic Stimulation
Andre Galenchik-Chan *1, Diane Chernoff *1, Wei Zhou 2, Timothy Q. Duong 3, Eric R. Muir 4
1Renaissance School of Medicine at Stony Brook University, 2Department of Radiology, University of Colorado School of Medicine, 3Department of Radiology, Montefiore-Einstein, 4Department of Radiology, School of Medicine, Stony Brook University

We have developed techniques for mapping the visual cortex function utilizing more of the visual field than is commonly used. This approach has the potential to enhance the evaluation of vision disorders and eye diseases.

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