S'identifier

New York University School of Medicine

34 ARTICLES PUBLISHED IN JoVE

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Neuroscience

SDS-PAGE/Immunoblot Detection of Aβ Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval
Rebecca F. Rosen 1, Yasushi Tomidokoro 2, Jorge A. Ghiso 3, Lary C. Walker 1,4
1Yerkes National Primate Research Center, Emory University, 2Department of Neurology, Institute of Clinical Medicine, Tsukuba University, 3Department of Pathology, New York University School of Medicine, 4Department of Neurology, Emory University

We describe a technique for the preparation of clarified human cortical homogenates, protein separation by SDS-PAGE, antigen retrieval and immunoblotting with an antibody to the Aβ peptide. Using this protocol, we consistently detect monomeric and multimeric Aβ in cortical tissue from humans with Alzheimer's pathology.

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Immunology and Infection

Retroviral Transduction of T-cell Receptors in Mouse T-cells
Shi Zhong 1, Karolina Malecek 1,2, Arianne Perez-Garcia 1, Michelle Krogsgaard 1
1NYU Cancer institute, New York University School of Medicine, 2Program in Structural Biology, New York University School of Medicine

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

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Neuroscience

Targeted Training of Ultrasonic Vocalizations in Aged and Parkinsonian Rats
Aaron M. Johnson 1,2, Emerald J. Doll 2, Laura M. Grant 2, Lauren Ringel 2, Jaime N. Shier 2, Michelle R. Ciucci 1,2
1Department of Surgery-Division of Otolaryngology, University of Wisconsin, 2Department of Communicative Disorders, University of Wisconsin

Voice disorders are debilitating in aging and Parkinson disease. The ultrasonic vocalizations of rats, also affected by these conditions, can be used to study these voice disorders, their neural substrates, and the nature of functional recovery with behavioral intervention.

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Biology

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin
Qing-Yun Tian 1, Yun-Peng Zhao 1, Chuan-ju Liu 1,2
1Department of Orthopaedic Surgery, NYU Hospital for Joint Diseases, 2Department of Cell Biology, New York University School of Medicine

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

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Immunology and Infection

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation
Mariko Kobayashi 1, Ju-Youn Kim 1, Vladimir Camarena 2, Pamela C. Roehm 3, Moses V. Chao 2,4,5,6,7, Angus C. Wilson 1, Ian Mohr 1
1Department of Microbiology, New York University School of Medicine, 2Molecular Neurobiology Program, Skirball Institute for Biomolecular Medicine, New York University School of Medicine, 3Department of Otolaryngology, New York University School of Medicine, 4Department of Cell Biology, New York University School of Medicine, 5Department of Physiology and Neuroscience, New York University School of Medicine, 6Department of Psychiatry, New York University School of Medicine, 7Center for Neural Science, New York University School of Medicine

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

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Immunology and Infection

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting
Dumizulu L. Tembo 1, Jacqui Montgomery 1, Alister G. Craig 2, Samuel C. Wassmer 3
1Malawi-Liverpool-Wellcome Trust Clinical Research Programme, 2Liverpool School of Tropical Medicine, 3Department of Microbiology, Division of Medical Parasitology, New York University School of Medicine

This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC.

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JoVE Journal

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice
Wen-Chi Chen *1, Sung-Hyun Park *1, Carol Hoffman 1, Cecil Philip 1, Linda Robinson 2, James West 2, Gabriele Grunig 1,3
1Department of Environmental Medicine, New York University School of Medicine, Tuxedo, 2Division of Allergy, Pulmonary, & Critical Care Medicine, Department of Medicine, Vanderbilt University Medical Center, 3Division of Pulmonary Medicine, New York University School of Medicine

A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies.

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Bioengineering

Skin Tattooing As A Novel Approach For DNA Vaccine Delivery
Yung-Nung Chiu 1, Jared M. Sampson 1, Xunqing Jiang 1, Susan B. Zolla-Pazner 2,3, Xiang-Peng Kong 1
1Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, 2Department of Pathology, New York University School of Medicine, 3Healthcare System, Veterans Affairs New York Harbor

Skin tattooing is a potent and safe way to delivery DNA vaccine intradermally. Here, a DNA plasmid encoding EGFP is delivered by tattooing to the skin of a laboratory mouse, and the expression of EGFP in the skin cells is then inspected by confocal microscopy.

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Biology

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization
Julie Chaumeil 1, Mariann Micsinai 1,2,3,4, Jane A. Skok 1
1Department of Pathology, New York University School of Medicine, 2New York University Center for Health Informatics and Bioinformatics, 3NYU Cancer Institute, 4Department of Pathology and Yale Cancer Center, Yale University School of Medicine

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

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Bioengineering

Establishment of a Surgically-induced Model in Mice to Investigate the Protective Role of Progranulin in Osteoarthritis
Yunpeng Zhao 1, Ben Liu 1, Chuan-ju Liu 2
1Department of Orthopaedic Surgery, NYU Hospital for Joint Diseases, 2Department of Orthopaedic Surgery, New York University Medical Center

We describe a protocol for the destabilization of the medial meniscus (DMM) model in mice, an effective tool for osteoarthritis (OA) research. In addition, we have demonstrated that deficiency of progranulincan exaggerate OA development and progression by using this model, indicating that progranulin plays a protective role in the pathogenesis of OA.

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Immunology and Infection

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1β in Human Monocyte-derived Dendritic Cells
Melissa V. Fernandez 1, Elizabeth A. Miller 2, Nina Bhardwaj 3
1Department of Pathology, New York University School of Medicine, 2Division of Infectious Diseases, Department of Medicine, Mount Sinai Medical Center, 3Division of Hematology and Oncology, Hess Center for Science and Medicine, Mount Sinai Medical Center

Dendritic cells (DCs) secrete IL-1β in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1β can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1β expression.

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Neuroscience

Subtype-selective Electroporation of Cortical Interneurons
Natalia V. De Marco Garcia 1,2, Gord Fishell 1
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.

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JoVE Journal

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes
Marianne Strazza 1, Inbar Azoulay-Alfaguter 1, Ariel Pedoeem 1, Adam Mor 1,2
1Department of Medicine, New York University School of Medicine, 2Departments of Pathology, New York University School of Medicine

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

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Developmental Biology

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells
Ieng Lam Lei 1, Lei Bu 2, Zhong Wang 1
1Cardiac Surgery, University of Michigan, 2Leon H Charney Division of Cardiology, New York University School of Medicine

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

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Medicine

MRI Mapping of Cerebrovascular Reactivity via Gas Inhalation Challenges
Hanzhang Lu 1, Peiying Liu 1, Uma Yezhuvath 1, Yamei Cheng 1, Olga Marshall 2, Yulin Ge 2
1Advanced Imaging Research Center, University of Texas Southwestern Medical Center, 2Center for Biomedical Imaging, Department of Radiology, New York University School of Medicine

Non-invasive imaging of the brain vasculature’s ability to dilate or constrict may allow a better understanding of cerebrovascular pathophysiology in various neurological diseases. The present report describes a reproducible and patient-comfortable protocol to perform vascular reactivity imaging in humans using magnetic resonance imaging (MRI).

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Neuroscience

Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue
Nicole Haack 1, Simone Durry 1, Karl W. Kafitz 1, Mitchell Chesler 2, Christine R. Rose 1
1Institute of Neurobiology, Heinrich Heine University Düsseldorf, 2Departments of Physiology and Neuroscience, New York University School of Medicine

We demonstrate the fabrication, calibration and properties of two types of ion-selective microelectrodes (double-barreled and concentric) for measurement of ion concentrations in brain tissue. These are then used in the mouse hippocampal slice preparation to show that excitatory activity changes both extracellular potassium and sodium concentrations.

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Immunology and Infection

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions
Marianne Strazza 1, Inbar Azoulay-Alfaguter 1, Michael Peled 1, Adam Mor 1,2
1Department of Medicine, New York University School of Medicine, 2Department of Pathology, New York University School of Medicine

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

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Biochemistry

Protein Complex Affinity Capture from Cryomilled Mammalian Cells
John LaCava 1,2, Hua Jiang 1, Michael P. Rout 1
1Laboratory of Cellular and Structural Biology, The Rockefeller University, 2Institute for Systems Genetics, Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

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Genetics

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format
Muhammad Khairul Ramlee 1, Jing Wang 1, Alice M. S. Cheung 1, Shang Li 1
1Cancer & Stem Cell Biology Programme, Duke-NUS Medical School

The genotyping technique described here, which couples fluorescent polymerase chain reaction (PCR) to capillary gel electrophoresis, allows for high-throughput genotyping of nuclease-mediated knockout clones. It circumvents limitations faced by other genotyping techniques and is more cost effective than sequencing methods.

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Neuroscience

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method
Thomas R. Groves 1,2,3, Jing Wang 1,2, Marjan Boerma 1,2, Antiño R. Allen 1,2,3
1Division of Radiation Health, University of Arkansas for Medical Sciences, 2Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, 3Neurobiology & Developmental Sciences, University of Arkansas for Medical Sciences

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

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Neuroscience

Real-time Iontophoresis with Tetramethylammonium to Quantify Volume Fraction and Tortuosity of Brain Extracellular Space
John Odackal *1, Robert Colbourn *2,3, Namrita Jain Odackal 4, Lian Tao 5, Charles Nicholson 5, Sabina Hrabetova 2
1Department of Medicine, University of Virginia, 2Department of Cell Biology, SUNY Downstate Medical Center, 3Neural and Behavioral Science Graduate Program, SUNY Downstate Medical Center, 4Division of Neonatology, University of Virginia, 5Department of Neuroscience and Physiology, New York University School of Medicine

This protocol describes real-time iontophoresis, a method that measures physical parameters of the extracellular space (ECS) of living brains. The diffusion of an inert molecule released into the ECS is used to calculate the ECS volume fraction and tortuosity. It is ideal for studying acute reversible changes to brain ECS.

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Immunology and Infection

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes
Michael Peled 1, Marianne Strazza 2, Adam Mor 2
1Pulmonary Department, The Chaim Sheba Medical Center, 2Columbia Center for Translational Immunology, Department of Medicine, Columbia University Medical Center

Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.

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Immunology and Infection

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model
Piul S. Rabbani 1, Salma A. Abdou 1, Darren L. Sultan 1, Jennifer Kwong 1, April Duckworth 1, Daniel J. Ceradini 1
1Hansjörg Wyss Department of Plastic Surgery, New York University School of Medicine

We describe a non-invasive in vivo imaging protocol that is streamlined and cost-effective, utilizing L-012, a chemiluminescent luminol-analog, to visualize and quantify reactive oxygen species (ROS) generated in a mouse excisional wound model.

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Neuroscience

Intracranial Pharmacotherapy and Pain Assays in Rodents
Erik Martinez 1, Haocheng Zhou 1, Jing Wang 1,2
1Department of Anesthesiology, Perioperative Care and Pain Medicine, New York University School of Medicine, 2Department of Neuroscience and Physiology, New York University School of Medicine

Here we present a protocol to perform intracranial pharmacological experiments followed by pain behavior assays in rodents. This protocol allows researchers to deliver molecular and cellular targets in the brain, for pharmacologic agents in the treatment of pain.

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Neuroscience

Bilateral Assessment of the Corticospinal Pathways of the Ankle Muscles Using Navigated Transcranial Magnetic Stimulation
Charalambos C. Charalambous 1,2, Jing Nong Liang 3,4, Steve A. Kautz 2,5, Mark S. George 5,6, Mark G. Bowden 2,5,7
1Department of Neurology, New York University School of Medicine, 2Department of Health Sciences and Research, Medical University of South Carolina, 3Department of Physical Therapy, University of Nevada Las Vegas, 4Department of Health Professions, Medical University of South Carolina, 5Ralph H. Johnson VA Medical Center, 6Department of Psychiatry, Medical University of South Carolina, 7Division of Physical Therapy, Medical University of South Carolina

The present protocol describes the simultaneous, bilateral assessment of the corticomotor response of the tibialis anterior and soleus during rest and tonic voluntary activation using a single pulse transcranial magnetic stimulation and neuronavigation system.

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Biochemistry

A Semi-Quantitative Drug Affinity Responsive Target Stability (DARTS) assay for studying Rapamycin/mTOR interaction
Chen Zhang *1, Min Cui *1, Yazhou Cui 1, Aubryanna Hettinghouse 1, Chuan-ju Liu 1,2
1Department of Orthopaedic Surgery, New York University Medical Center, 2Department of Cell Biology, New York University School of Medicine

In this study, we enhanced the data analysis capabilities of the DARTS experiment by monitoring the changes in protein stability and estimating the affinity of protein-ligand interactions. The interactions can be plotted into two curves: a proteolytic curve and a dose-dependence curve. We have used mTOR-rapamycin interaction as an exemplary case.

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Medicine

The Lower Body Positive Pressure Treadmill for Knee Osteoarthritis Rehabilitation
Junjie Liang *1,2, Yuanyuan Guo *1,2, Yuxin Zheng 1,2, Shijuan Lang 1, Hongxin Chen 1,2, Yaoyao You 1,2, Bryan O’Young 3,4, Haining Ou *1,2, Qiang Lin *1,2
1Department of Rehabilitation, The Fifth Affiliated Hospital of Guangzhou Medical University, 2Experiment Education Model Center of Rehabilitation Medicine, Guangzhou Medical University, 3Department of Physical Medicine and Rehabilitation, Geisinger Health System, 4Department of Rehabilitation Medicine, New York University School of Medicine

Here, based on a clinician’s point-of-view, we propose a two-model lower body positive pressure (LBPP) protocol (walking and squatting models) in addition to a clinical, functional assessment methodology, including details for further encouragement of the development of non-drug surgical intervention strategies in knee osteoarthritis patients. However, we only present the effect of LBPP training in improvement of pain and knee function in one patient through three-dimensional gait analysis. The exact, long-term effects of this approach should be explored in future studies.

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Genetics

A Method to Study de novo Formation of Chromatin Domains
Ozgur Oksuz 1,2,3, Danny Reinberg 1,2
1Howard Hughes Medical Institute, New York University School of Medicine, 2Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, 3Whitehead Institute for Biomedical Research

This method is designed to follow formation of PRC2-mediated chromatin domains in cell lines, and the method can be adapted to many other systems.

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Biology

Quantification of Proliferative and Dead Cells in Enteroids
Hua-Shan Li *1, Shao-Fang Xu *1, Jian-Ying Sheng *1, Zhi-Hui Jiang 1, Jing Wang 1, Ning Ding 1, Tao Wang 1, Matthew A. Odenwald 2, Jerrold R. Turner 2,3, Wei-Qi He 1, Hong Xu 1, Juan-Min Zha 1
1Jiangsu Key Laboratory of Neuropsychiatric Diseases and Cambridge-Suda (CAM-SU) Genomic Resource Center, Medical College of Soochow University, Department of Oncology, The First Affiliated Hospital of Soochow University, 2Department of Pathology, University of Chicago, 3Department of Pathology, Brigham and Women's Hospital–Harvard Medical School

The presented protocol uses flow cytometry to quantify the number of proliferating and dead cells in cultured mouse enteroids. This method is helpful to evaluate the effects of drug treatment on organoid proliferation and survival.

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Immunology and Infection

Generation of Human Regulatory T Cell Clones
Johannes Nowatzky 1, Olivier Manches 2,3
1Department of Medicine, Division of Rheumatology, New York University School of Medicine, 2Immunobiology and Immunotherapy in Chronic Diseases, Institute for Advanced Biosciences, Université Grenoble Alpes, Inserm U1209/CNRS UMR 5309, 3Etablissement Français du Sang Auvergne-Rhône-Alpes

This protocol describes the cloning and expansion of human regulatory T cells for the generation of ultra-high purity viable human Treg with stable demethylation at the Treg-specific demethylated region (TSDR) and Treg-specific phenotypic features.

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Neuroscience

Preparation of the Rat Vocal Fold for Neuromuscular Analyses
Charles Lenell 1,2, Adrianna C. Shembel 1, Aaron M. Johnson 1
1NYU Voice Center, Department of Otolaryngology-Head & Neck Surgery, New York University School of Medicine, 2Communicative Sciences and Disorders, New York University

This protocol describes methods used to prepare rat vocal folds for histochemical neuromuscular study.

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Genetics

Embryo Injections for CRISPR-Mediated Mutagenesis in the Ant Harpegnathos saltator
Kayli Sieber 1, Maya Saar 1, Comzit Opachaloemphan 2, Matthew Gallitto 3, Huan Yang 2, Hua Yan 1,4
1Department of Biology, University of Florida, 2Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, 3Department of Radiation Oncology, Columbia University Medical Center, 4Center for Smell and Taste, University of Florida

Many characteristics of insect eusociality rely on within-colony communication and division of labor. Genetic manipulation of key regulatory genes in ant embryos via microinjection and CRISPR-mediated mutagenesis provides insights into the nature of altruistic behavior in eusocial insects.

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Biology

Laser Micro-Irradiation to Study DNA Recruitment During S Phase
Bearach Miwatani-Minter 1,2, Gergely Rona 1,2,3
1Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, 2Laura and Isaac Perlmutter Cancer Center, New York University School of Medicine, 3Howard Hughes Medical Institute, New York University School of Medicine

This protocol describes a non-invasive method to efficiently identify S-phase cells for downstream microscopy studies, such as measuring DNA repair protein recruitment by laser micro-irradiation.

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Neuroscience

PyDesigner v1.0: A Pythonic Implementation of the DESIGNER Pipeline for Diffusion Magnetic Resonance Imaging
Siddhartha Dhiman *1, Reyna E. Hickey *2, Kathryn E. Thorn *2, Hunter G. Moss 1,3, Emilie T. McKinnon 1,3, Vitria Adisetiyo 1, Benjamin Ades-Aron 4, Jens H. Jensen 1,3,5, Andreana Benitez 2,3
1Department of Neuroscience, Medical University of South Carolina, 2Department of Neurology, Medical University of South Carolina, 3Center for Biomedical Imaging, Medical University of South Carolina, 4Center for Biomedical Imaging, Department of Radiology, New York University School of Medicine, 5Department of Radiology and Radiological Science, Medical University of South Carolina

Here we present PyDesigner, a Python-based diffusion magnetic resonance imaging (dMRI) processing pipeline capable of correcting for typical dMRI artifacts and producing diffusion tensor imaging (DTI), diffusion kurtosis imaging (DKI), fiber ball imaging (FBI), microstructure modeling (white matter integrity [WMTI] and fiber ball white matter [FBWM]), and tractography outputs.

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