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University of Michigan Medical School

24 ARTICLES PUBLISHED IN JoVE

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Biology

Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster
Martin J. Tinkerhess 1, Sara Ginzberg 1, Nicole Piazza 1, Robert J. Wessells 1
1Department of Geriatric Medicine, University of Michigan Medical School

We describe the first endurance training protocol for an important genetic model species, Drosophila melanogaster, and outline several assays to chart improvements in mobility following training.

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Biology

Force Measurement During Contraction to Assess Muscle Function in Zebrafish Larvae
Darcée D. Sloboda 1, Dennis R. Claflin 1,2, James J. Dowling 3, Susan V. Brooks 1,4
1Department of Biomedical Engineering, University of Michigan , 2Department of Surgery, Section of Plastic Surgery, University of Michigan , 3Departments of Pediatrics and Neurology, University of Michigan , 4Department of Molecular and Integrative Physiology, University of Michigan

Force measurements can be used to demonstrate changes in muscle function due to development, injury, disease, treatment or chemical toxicity. In this video, we demonstrate a method to measure force during a maximal contraction of zebrafish larval trunk muscle.

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Medicine

Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma
Anda-Alexandra Calinescu 1, Felipe Javier Núñez 1, Carl Koschmann 2, Bradley L. Kolb 1, Pedro R. Lowenstein 1,3, Maria G. Castro 1,3
1Department of Neurosurgery, University of Michigan School of Medicine, 2Department of Pediatrics, Division of Hematology-Oncology, University of Michigan School of Medicine, 3Department of Cell and Developmental Biology, University of Michigan

Here we describe an efficient and versatile protocol to induce, monitor and analyze novel glioblastomas (GBM) using transposon DNA injected into the ventricles of neonatal mice. Cells of the subventricular zone, which take up the plasmid, transform, proliferate and generate tumors with histo-pathological characteristics of human GBM.

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Bioengineering

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers
Stuart M. Roche 1, Jonathan P. Gumucio 1,2, Susan V. Brooks 2,3, Christopher L. Mendias 1,2, Dennis R. Claflin 3,4
1Department of Orthopaedic Surgery, University of Michigan Medical School, 2Department of Molecular & Integrative Physiology, University of Michigan Medical School, 3Department of Biomedical Engineering, University of Michigan Medical School, 4Department of Surgery, Section of Plastic Surgery, University of Michigan Medical School

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. In this article we outline a valid and reliable technique to prepare and test permeabilized skeletal muscle fibers in vitro.

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Medicine

Direct Mouse Trauma/Burn Model of Heterotopic Ossification
Jonathan R. Peterson 1, Shailesh Agarwal 1, R. Cameron Brownley 1, Shawn J. Loder 1, Kavitha Ranganathan 1, Paul S. Cederna 1, Yuji Mishina 2, Stewart C. Wang 1, Benjamin Levi 1
1Department of Surgery, University of Michigan Medical School, 2Department of Biologic and Materials Sciences, University of Michigan School of Dentistry

An Achilles tenotomy and burn injury model of heterotopic ossification allows for the reliable study of trauma induced ectopic bone formation without the application of exogenous factors.

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Neuroscience

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers
Hussein S. Hamid 1, John M. Hayes 2, Eva L. Feldman 2, Stephen I. Lentz 3
1University of Michigan Medical School, 2Department of Neurology, University of Michigan, 3Department of Internal Medicine, University of Michigan

This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.

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Immunology and Infection

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells
Gregory J. Baker 1,2, Maria G. Castro 1,2, Pedro R. Lowenstein 1,2
1Department of Neurosurgery, University of Michigan, 2Department of Cell and Developmental Biology, University of Michigan

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

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Immunology and Infection

Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle (GUV) Membranes
Balaji Olety 1, Sarah L. Veatch 2, Akira Ono 1
1Department of Microbiology and Immunology, University of Michigan Medical School, 2Department of Biophysics, University of Michigan

We illustrate here an in vitro membrane binding assay in which interactions between HIV-1 Gag and lipid membranes are visually analyzed using YFP-tagged Gag synthesized in a wheat germ-based in vitro translation system and GUVs prepared by an electroformation technique.

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Developmental Biology

Stimulation of Notch Signaling in Mouse Osteoclast Precursors
Gurpreet Kaur 1,2, Jaimo Ahn 1, Kurt D. Hankenson 1,3, Jason W. Ashley 1,4
1Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, 2Department of Biological Sciences, University of the Sciences, 3The Department of Small Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, 4Department of Biology, College of Science, Technology, Engineering, & Mathematics, Eastern Washington University

Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

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Biochemistry

Investigating Protein Sequence-structure-dynamics Relationships with Bio3D-web
Shashank Jariwala *1, Lars Skjærven *2, Xin-Qiu Yao 1, Barry J. Grant 1
1Department of Computational Medicine and Bioinformatics, University of Michigan Medical School, 2Department of Biomedicine, University of Bergen

A protocol for the online investigation of protein sequence-structure-dynamics relationships using Bio3D-web is presented.

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Medicine

Comparing the Effects of Electronic Cigarette Vapor and Cigarette Smoke in a Novel In Vivo Exposure System
Anthony N. Hage 1, Will Krause 1, Angela Mathues 1, Luke Krasner 2, Seth Kasten 1, Jonathan L. Eliason 1,3, Abhijit Ghosh 1
1Jobst Vascular Research Laboratory, University of Michigan Medical School, 2Department of Engineering, Purdue University, 3Department of Surgery, Section of Vascular Surgery, University of Michigan Health System

This protocol describes a method for exposing rodents to electronic cigarette vapor (E-vapor) and cigarette smoke. Exposure chambers are constructed by modifying anesthesia chambers with an automated pumping system that delivers E-vapor or cigarette smoke to rodents. This system can easily be modified to accommodate many experimental endpoints.

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Cancer Research

Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres
Flor M. Mendez 1, Felipe J. Núñez 1,2, Rocío I. Zorrilla-Veloz 3,4, Pedro R. Lowenstein 1,2, Maria G. Castro 1,2
1Department of Cell and Developmental Biology, University of Michigan Medical School, 2Department of Neurosurgery, University of Michigan Medical School, 3Cancer Research Summer Internship Program (CARSIP), Cancer Biology Program, University of Michigan Medical School, 4Department of Biology, University of Puerto Rico-Río Piedras Campus

Epigenetic mechanisms are frequently altered in glioma. Chromatin immunoprecipitation could be used to study the consequences of genetic alterations in glioma that result from changes in histone modifications which regulate chromatin structure and gene transcription. This protocol describes native chromatin immunoprecipitation on murine brain tumor neurospheres.

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Medicine

Fracture Apparatus Design and Protocol Optimization for Closed-stabilized Fractures in Rodents
Robert L Zondervan 1,2, Mitch Vorce 3, Nick Servadio 4, Kurt D. Hankenson 2
1College of Osteopathic Medicine, Michigan State University, 2Department of Orthopaedic Surgery, University of Michigan Medical School, 3Lymann Briggs College, Michigan State University, 4College of Engineering, Michigan State University

The goal of the protocol is to optimize the fracture generation parameters to yield consistent fractures. This protocol accounts for the variations in bone size and morphology that may exist between animals. Additionally, a cost-effective, adjustable fracture apparatus is described.

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Cancer Research

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures
Felipe J. Núñez 1,2, Flor M. Mendez 2, Maria B. Garcia-Fabiani 1,2, Joaquín Pardo 1,3, Marta Edwards 1,2, Pedro R. Lowenstein 1,2, Maria G. Castro 1,2
1Department of Neurosurgery, University of Michigan Medical School, 2Department of Cell & Developmental Biology, University of Michigan, 3INIBIOLP, Histology B-Pathology B, School of Medicine, UNLP

Neurospheres grown as 3D cultures constitute a powerful tool to study glioma biology. Here we present a protocol to perform immunohistochemistry while maintaining the 3D structure of glioma neurospheres through paraffin embedding. This method enables the characterization of glioma neurosphere properties such as stemness and neural differentiation.

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Medicine

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer
Ligia B. Schmitd 1, Min Liu 1, Christina S. Scanlon 1, Rajat Banerjee 1, Nisha J. D’Silva 1,2
1Periodontics and Oral Medicine, University of Michigan School of Dentistry, 2Pathology, University of Michigan Medical School

Perineural invasion is an aggressive phenotype for head and neck squamous cell carcinomas and other tumors. The chick chorioallantoic membrane model has been used for studying angiogenesis, cancer invasion, and metastasis. Here we demonstrate how this model can be utilized to assess perineural invasion in vivo.

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Biology

Identification and Dissection of Diverse Mouse Adipose Depots
Devika P. Bagchi 1, Ormond A. MacDougald 1
1Department of Molecular & Integrative Physiology, University of Michigan Medical School

Adipocytes exist in discrete depots and have diverse roles within their unique microenvironments. As regional differences in adipocyte character and function are uncovered, standardized identification and isolation of depots is crucial for advancement of the field. Herein, we present a detailed protocol for the excision of various mouse adipose depots.

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Bioengineering

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk
Carmen G. Flesher 1, Nicki A. Baker 1, Clarissa Strieder-Barboza 1,2, Dominic Polsinelli 5, Phillip J. Webster 1,2, Oliver A. Varban 1, Carey N. Lumeng 2,3,4, Robert W. O'Rourke 1,6
1Department of Surgery, University of Michigan Medical School, 2Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, 3Graduate Program in Immunology, University of Michigan Medical School, 4Graduate Program in Cellular and Molecular Biology, University of Michigan Medical School, 5Undergraduate Research Opportunity Program, University of Michigan, 6Department of Surgery, Ann Arbor Veterans Affairs Healthcare System

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

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Neuroscience

Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion
Andrea Comba 1,2,4, Patrick J. Dunn 1,2,4, Phillip E. Kish 1,3, Padma Kadiyala 1,2,4, Alon Kahana 3, Maria G. Castro 1,2,4, Pedro R. Lowenstein 1,2,4
1Dept. of Neurosurgery, University of Michigan Medical School, 2Dept. of Cell and Developmental Biology, University of Michigan Medical School, 3Dept. of Ophthalmology & Visual Science, University of Michigan Medical School, 4Rogel Cancer Center, University of Michigan Medical School

Laser microdissection (LMD) is a sensitive and highly reproducible technique that can be used to uncover pathways that mediate glioma heterogeneity and invasion. Here, we describe an optimized protocol to isolate discrete areas from glioma tissue using laser LMD followed by transcriptomic analysis.

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Cancer Research

Quantifying the Brain Metastatic Tumor Micro-Environment using an Organ-On-A Chip 3D Model, Machine Learning, and Confocal Tomography
C. Ryan Oliver *1,2, Trisha M. Westerhof *1,2, Maria G. Castro 2,3,4, Sofia D. Merajver 1,2
1Department of Internal Medicine, University of Michigan Ann Arbor, 2Rogel Cancer Center, University of Michigan Ann Arbor, 3Department of Neurosurgery, University of Michigan Ann Arbor, 4Department of Cell and Developmental Biology, University of Michigan Ann Arbor

Here, we present a protocol for preparing and culturing a blood brain barrier metastatic tumor micro-environment and then quantifying its state using confocal imaging and artificial intelligence (machine learning).

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Biology

Production of IgG Fusion Proteins Transiently Expressed in Nicotiana benthamiana
Aigerim S. Kamzina *1,2,3, Michelle P. DiPalma *1,2,3, Joseph G. L. Hunter 1,2,3, Andrew G. Diamos 1,2,4, Boyd Armer 2,3, Tsafrir S. Mor 1,2,3, Hugh S. Mason 1,2,3
1Center for Immunotherapy, Vaccines and Virotherapy, The Biodesign Institute, Arizona State University, 2School of Life Sciences, Arizona State University, 3Molecular Biosciences/Biotechnology Undergraduate Program, Arizona State University, 4Department of Microbiology and Immunology, University of Michigan Medical School

We describe here a simple method for expression, extraction, and purification of recombinant human IgG fused to GFP in Nicotiana benthamiana. This protocol can be extended to purification and visualization of numerous proteins that utilize column chromatography. Moreover, the protocol is adaptable to the in-person and virtual college teaching laboratory, providing project-based exploration.

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Bioengineering

The Muscle Cuff Regenerative Peripheral Nerve Interface for the Amplification of Intact Peripheral Nerve Signals
Shelby R. Svientek 1, Justin P. Wisely 1, Amir Dehdashtian 1, Jarred V. Bratley 1, Paul S. Cederna 1,2, Stephen W. P. Kemp 1,2
1Department of Surgery, Section of Plastic Surgery, The University of Michigan Health System, 2Department of Biomedical Engineering, The University of Michigan, Ann Arbor

This manuscript provides an innovative method for developing a biologic peripheral nerve interface termed the Muscle Cuff Regenerative Peripheral Nerve Interface (MC-RPNI). This surgical construct can amplify its associated peripheral nerve's motor efferent signals to facilitate accurate detection of motor intent and the potential control of exoskeleton devices.

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Biochemistry

Detection and Quantification of Calcitonin Gene-Related Peptide (CGRP) in Human Plasma Using a Modified Enzyme-Linked Immunosorbent Assay
Pavan S. Krishnan 1,2, Fernando T. Zamuner 1,3, Carolyn M. Jenks 1, Johnny Y. Xie 4, Lisa Zhang 5, Mohammed Lehar 1, Neal S. Fedarko 6, Mariana Brait 1,3, John P. Carey 1
1Department of Otolaryngology-Head & Neck Surgery, Johns Hopkins University School of Medicine, 2Virginia Commonwealth University School of Medicine, 3Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, 4Department of Otolaryngology-Head and Neck Surgery, University of Michigan Medical School, 5Department of Otolaryngology-Head and Neck Surgery, The Ohio State University College of Medicine, 6ICTR Clinical Research Core Laboratory, Johns Hopkins University School of Medicine

Published data pertaining to calcitonin gene-related peptide (CGRP) concentrations in human plasma are inconsistent. These inconsistencies may be due to the lack of a standardized, validated methodology to quantify this neuropeptide. Here, we describe a validated enzyme-linked immunosorbent assay (ELISA) protocol to purify and quantify CGRP in human plasma.

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Biology

Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic β-Cells
Elena Levi-D’Ancona 1,2, Vaibhav Sidarala 1, Scott A. Soleimanpour 1,3,4
1Department of Internal Medicine, Division of Metabolism, Endocrinology and Diabetes, University of Michigan, Ann Arbor, 2Graduate Program in Immunology, University of Michigan Medical School, 3Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, 4VA Ann Arbor Healthcare System

This protocol outlines two methods for the quantitative analysis of mitophagy in pancreatic β-cells: first, a combination of cell-permeable mitochondria-specific dyes, and second, a genetically encoded mitophagy reporter. These two techniques are complementary and can be deployed based on specific needs, allowing for flexibility and precision in quantitatively addressing mitochondrial quality control.

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Neuroscience

Development of a Low-cost Epimysial Electromyography Electrode: A Simplified Workflow for Fabrication and Testing
Luke Stoneback *1, Genaro D. Fullano *2, McKenzie S. White 1, Sairub Naaz 1, Lindsey K. Lepley 1
1School of Kinesiology, University of Michigan, Ann Arbor, 2University of Michigan Medical School

Our purpose was to provide an updated, easy-to-follow guide on the fabrication and testing of epimysial electromyography electrodes. To that end, we provide instructions for material sourcing and a detailed walkthrough of the fabrication and testing process.

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