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Perelman School of Medicine, University of Pennsylvania

12 ARTICLES PUBLISHED IN JoVE

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Biology

Isolation and Primary Culture of Rat Hepatic Cells
Ling Shen 1, Allix Hillebrand 2, David Q.-H. Wang 3, Min Liu 1
1Department of Pathology and Laboratory Medicine, University of Cincinnati College of Medicine, 2American University in Washington, D.C., 3Department of Internal Medicine, Saint Louis University School of Medicine

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 × 108 cells per preparation with cell viability between 88 ~ 96%.

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Biology

Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies
Catherine Moorwood 1, Min Liu 2, Zuozhen Tian 2, Elisabeth R. Barton 3
1Department of Anatomy and Cell Biology, School of Dental Medicine, University of Pennsylvania, 2Department of Physiology, Perelman School of Medicine, University of Pennsylvania, 3Department of Anatomy and Cell Biology, School of Dental Medicine, School of Dental Medicine, University of Pennsylvania

Muscle function measurements contribute to the evaluation of potential therapeutics for muscle pathology, as well as to the determination of mechanisms underlying physiology of this tissue. We will demonstrate the preparation of the extensor digitorum longus and diaphragm muscles for functional testing. Protocols for isometric and eccentric contractions will be shown, as well as differences in results between dystrophic muscles, representing a pathological state, and wildtype muscles.

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Biology

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)
Sandra Deliard 1, Jianhua Zhao 1, Qianghua Xia 1, Struan F.A. Grant 1,2
1Division of Human Genetics, Children's Hospital of Philadelphia Research Institute, 2Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

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JoVE Core

Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention
Hilary S. McCarren 1,2,4, Jason T. Moore 1,3,4, Max B. Kelz 1,2,3,4
1Department of Anesthesiology and Critical Care, Perelman School of Medicine, University of Pennsylvania, 2Department of Pharmacology, Perelman School of Medicine, University of Pennsylvania, 3Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania, 4Center for Sleep and Circadian Neurobiology, Perelman School of Medicine, University of Pennsylvania

Loss of the righting reflex has long served as a standard behavioral surrogate for unconsciousness, also called hypnosis, in laboratory animals. Alterations in volatile anesthetic sensitivity caused by pharmacological interventions can be detected with a carefully controlled high-throughput assessment system, which may be adapted for delivery of any inhaled therapeutic.

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Cancer Research

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors
Alan J. Fox 1, Matthew C. Hiemenz 1, David B. Lieberman 1, Shrey Sukhadia 1, Barnett Li 1, Joseph Grubb 1, Patrick Candrea 1, Karthik Ganapathy 1, Jianhua Zhao 1, David Roth 1, Evan Alley 2,3, Alison Loren 2,3, Jennifer J. D. Morrissette 1
1Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, 2Division of Hematology/Oncology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, 3Abramson Cancer Center

This manuscript describes clinical protocols for two next-generation sequencing panels. One panel interrogates hematologic malignancies while the other panel targets genes commonly mutated in solid tumors. Molecular classification of driver mutations in human malignancies offers valuable prognostic and predictive information.

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Neuroscience

High-density Electroencephalographic Acquisition in a Rodent Model Using Low-cost and Open-source Resources
Andrzej Z. Wasilczuk 1, Alexander Proekt 1, Max B. Kelz 1, Andrew R. McKinstry-Wu 1
1Department of Anesthesiology and Critical Care, Perelman School of Medicine, University of Pennsylvania

Instructions for the low-cost construction and surgical implantation of a chronic transcranial high-density electroencephalographic montage into mice are provided. Signal recording, extraction, and processing techniques are also described.

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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

A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells
Xiongwei Cai 1,2,3, Yi Zheng 1, Nancy A. Speck 2
1Division of Experimental Hematology, Cincinnati Children's Hospital Medical Center, 2Department of Cell and Developmental Biology, Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, 3Department of Obstetrics and Gynecology, Southwest Hospital, Third Military Medical University

A standard Western blotting protocol was optimized for analyzing as few as 500 hematopoietic stem or progenitor cells. Optimization involves careful handling of the cell sample, limiting transfers between tubes, and directly lysing the cells in Laemmli sample buffer.

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Immunology and Infection

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue
Maria Steblyanko 1, Nadia Anikeeva 1, Marcus Buggert 2,3, Michael R. Betts 2, Yuri Sykulev 4
1Department of Microbiology and Immunology, Thomas Jefferson University, 2Department of Microbiology and Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, 3Department of Medicine Huddinge, Karolinska Institutet, Karolinska University Hospital Huddinge, 4Departments of Microbiology and Immunology and Medical Oncology, Sidney Kimmel Cancer Center, Thomas Jefferson University

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

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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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Biology

Rapid Lipid Droplet Isolation Protocol Using a Well-established Organelle Isolation Kit
Jascha Brettschneider *1, Jason M. Correnti *1, Chelsea Lin 1, Bianca Williams 1, Amanke Oranu 1, Amy Kuriakose 1, Dru McIver-Jenkins 1, Abigail Haba 1, Isabelle Kaneza 1, Sookyoung Jeon 1, Eleonora Scorletti 1, Rotonya M. Carr 1
1Division of Gastroenterology, Perelman School of Medicine, University of Pennsylvania

This protocol establishes a novel method of lipid droplet isolation and purification from mouse livers, using a well-established endoplasmic reticulum isolation kit.

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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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