University of Michigan Medical School 18 articles published in JoVE Biology Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic β-Cells Elena Levi-D’Ancona1,2, Vaibhav Sidarala1, Scott A. Soleimanpour1,3,4 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. Biochemistry Detection and Quantification of Calcitonin Gene-Related Peptide (CGRP) in Human Plasma Using a Modified Enzyme-Linked Immunosorbent Assay Pavan S. Krishnan1,2, Fernando T. Zamuner1,3, Carolyn M. Jenks1, Johnny Y. Xie4, Lisa Zhang5, Mohammed Lehar1, Neal S. Fedarko6, Mariana Brait1,3, John P. Carey1 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. 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. Hunter1,2,3, Andrew G. Diamos1,2,4, Boyd Armer2,3, Tsafrir S. Mor1,2,3, Hugh S. Mason1,2,3 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. Neuroscience Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion Andrea Comba1,2,4, Patrick J. Dunn1,2,4, Phillip E. Kish1,3, Padma Kadiyala1,2,4, Alon Kahana3, Maria G. Castro1,2,4, Pedro R. Lowenstein1,2,4 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. Bioengineering A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk Carmen G. Flesher1, Nicki A. Baker1, Clarissa Strieder-Barboza1,2, Dominic Polsinelli5, Phillip J. Webster1,2, Oliver A. Varban1, Carey N. Lumeng2,3,4, Robert W. O'Rourke1,6 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. Biology Identification and Dissection of Diverse Mouse Adipose Depots Devika P. Bagchi1, Ormond A. MacDougald1 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. Medicine The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer Ligia B. Schmitd1, Min Liu1, Christina S. Scanlon1, Rajat Banerjee1, Nisha J. D’Silva1,2 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. Neuroscience Monitoring Neuronal Survival via Longitudinal Fluorescence Microscopy Kaitlin Weskamp*1,2, Nathaniel Safren*1, Roberto Miguez1, Sami Barmada1,2 1Department of Neurology, University of Michigan School of Medicine, 2Neuroscience Graduate Program, University of Michigan School of Medicine Here, we present a protocol to monitor survival on a single-cell basis and identify variables that significantly predict cell death. Cancer Research Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures Felipe J. Núñez1,2, Flor M. Mendez2, Maria B. Garcia-Fabiani1,2, Joaquín Pardo1,3, Marta Edwards1,2, Pedro R. Lowenstein1,2, Maria G. Castro1,2 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. Medicine Fracture Apparatus Design and Protocol Optimization for Closed-stabilized Fractures in Rodents Robert L Zondervan1,2, Mitch Vorce3, Nick Servadio4, Kurt D. Hankenson2 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. Cancer Research Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts Tina T. Thomas1, Sahiti Chukkapalli1, Raelene A. Van Noord1, Melanie Krook2, Mark J. Hoenerhoff3, Jonathan R. Dillman4, Elizabeth R. Lawlor2,5, Valerie P. Opipari5, Erika A. Newman1 1Departments of Surgery, C.S Mott Children's and Women's Hospital, The University of Michigan Medical School, 2Departments of Pathology, C.S Mott Children's and Women's Hospital, The University of Michigan Medical School, 3Unit for Laboratory Animal Medicine, The University of Michigan Medical School, 4Departments of Radiology, C.S Mott Children's and Women's Hospital, The University of Michigan Medical School, 5Departments of Pediatrics, C.S Mott Children's and Women's Hospital, The University of Michigan Medical School Here, we present a protocol to utilize ultrasound-guided injection of neuroblastoma (NB) and Ewing's sarcoma (ES) cells (established cell lines and patient-derived tumor cells) at biologically relevant sites to create reliable preclinical models for cancer research. Cancer Research Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres Flor M. Mendez1, Felipe J. Núñez1,2, Rocío I. Zorrilla-Veloz3,4, Pedro R. Lowenstein1,2, Maria G. Castro1,2 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. Neuroscience Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers Hussein S. Hamid1, John M. Hayes2, Eva L. Feldman2, Stephen I. Lentz3 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. Biochemistry Investigating Protein Sequence-structure-dynamics Relationships with Bio3D-web Shashank Jariwala*1, Lars Skjærven*2, Xin-Qiu Yao1, Barry J. Grant1 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. Medicine Comparing the Effects of Electronic Cigarette Vapor and Cigarette Smoke in a Novel In Vivo Exposure System Anthony N. Hage1, Will Krause1, Angela Mathues1, Luke Krasner2, Seth Kasten1, Jonathan L. Eliason1,3, Abhijit Ghosh1 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. Immunology and Infection Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle (GUV) Membranes Balaji Olety1, Sarah L. Veatch2, Akira Ono1 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. Medicine Direct Mouse Trauma/Burn Model of Heterotopic Ossification Jonathan R. Peterson1, Shailesh Agarwal1, R. Cameron Brownley1, Shawn J. Loder1, Kavitha Ranganathan1, Paul S. Cederna1, Yuji Mishina2, Stewart C. Wang1, Benjamin Levi1 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. Bioengineering Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers Stuart M. Roche1, Jonathan P. Gumucio1,2, Susan V. Brooks2,3, Christopher L. Mendias1,2, Dennis R. Claflin3,4 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.
