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University of Kansas Medical Center

17 ARTICLES PUBLISHED IN JoVE

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Medicine

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice
Nikki Cheng 1, Diana L. Lambert 1
1Department of Pathology, University of Kansas Medical Center

In this report, we demonstrate a system to isolate and culture donor cells from the mouse mammary gland, and orthotopically transplant these cells in recipient mice to analyze stromal: epithelial interactions during mammary tumor development.

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Medicine

Assessment of Perigenital Sensitivity and Prostatic Mast Cell Activation in a Mouse Model of Neonatal Maternal Separation
Isabella M. Fuentes 1, Angela N. Pierce 1, Pierce T. O'Neil 1, Julie A. Christianson 1
1Anatomy and Cell Biology, University of Kansas Medical Center

We are measuring perigenital mechanical sensitivity and mast cell activation in the prostate of male C57BL/6 mice that underwent an early life stress paradigm – neonatal maternal separation, in order to induce a preclinical model of chronic prostatitis/chronic pelvic pain syndrome.

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Neuroscience

Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction
Mychal S. Grames 1, Kasey L. Jackson 1, Robert D. Dayton 1, John A. Stanford 2, Ronald L. Klein 1
1Department of Pharmacology, Toxicology, and Neuroscience, Louisiana State University Health Sciences Center, 2Department of Molecular & Integrative Physiology, University of Kansas Medical Center

Methods for a wide-scale central nervous system gene delivery in the rat are covered. In this example, the purpose is to mimic a disease that affects the entire spinal cord. The widespread transduction can be used to deliver a therapeutic protein to the CNS from a one-time, peripheral administration.

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Bioengineering

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering
Christopher A. Neal 1, Jennifer G. Nelson-Brantley 1, Michael S. Detamore 2, Hinrich Staecker 1, Adam J. Mellott 3
1Department of Otolaryngology, University of Kansas Medical Center, 2Stephenson School of Biomedical Engineering, University of Oklahoma, 3Department of Plastic Surgery, University of Kansas Medical Center

The goal of this protocol is to demonstrate an effective method to decellularize and decalcify mouse cochleae for utilization as scaffolds for tissue engineering applications.

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Biochemistry

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy
Alexandra J. Machen 1, Pierce T. O'Neil 1, Bradley L. Pentelute 2, Maria T. Villar 1, Antonio Artigues 1, Mark T. Fisher 1
1Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, 2Department of Chemistry, Massachusetts Institute of Technology

Here we present a protocol to monitor the assembly and disassembly of the anthrax toxin using biolayer interferometry (BLI). Following assembly/disassembly on the biosensor surface, the large protein complexes are released from the surface for visualization and identification of components of the complexes using electron microscopy and mass spectrometry, respectively.

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Immunology and Infection

Induction and Scoring of Graft-Versus-Host Disease in a Xenogeneic Murine Model and Quantification of Human T Cells in Mouse Tissues using Digital PCR
Amara Seng 1, Mary A. Markiewicz 1
1Department of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center

Here, we present a protocol to induce and score disease in a xenogeneic graft-versus-host disease (xenoGVHD) model. xenoGVHD provides an in vivo model to study immunosuppression of human T cells. Additionally, we describe how to detect human T cells in tissues with digital PCR as a tool to quantify immunosuppression.

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Medicine

Imaging Features of Systemic Sclerosis-Associated Interstitial Lung Disease
Jonathan H. Chung 1, Christopher M. Walker 2, Stephen Hobbs 3
1Department of Radiology, University of Chicago Medicine, 2Department of Radiology, University of Kansas Medical Center, 3Department of Radiology, University of Kentucky

Here, we present practical recommendations for performing thoracic high-resolution computed tomography for diagnosing and assessing systemic sclerosis-related interstitial lung disease.

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Neuroscience

Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain
Prabhakar Singh 1, David W. Peng 1, William Z. Suo 1,2,3,4
1Laboratory for Alzheimer's Disease and Aging Research, Kansas City Veterans Affairs Medical Center, 2Department of Neurology, University of Kansas Medical Center, 3Department of Molecular and Integrative Physiology, University of Kansas Medical Center, 4The University of Kansas Alzheimer's Disease Center

Neuronal fiber length within a three-dimensional structure of a brain region is a reliable parameter to quantify specific neuronal structural integrity or degeneration. This article details a stereological quantification method to measure cholinergic fiber length within the nucleus basalis of Meynert in mice as an example.

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Neuroscience

Use of Capillary Electrophoresis Immunoassay to Search for Potential Biomarkers of Amyotrophic Lateral Sclerosis in Human Platelets
Jessica M. Sage 1, LaSharice Hall 2, April McVey 3, Richard J. Barohn 3, Jeffrey M. Statland 3, Omar Jawdat 3, Mazen M. Dimachkie 3, Abdulbaki Agbas 1
1Department of Basic Sciences, Kansas City University of Medicine and Biosciences, 2Rice University School of Medicine, 3University of Kansas Medical Center

Blood-based biomarkers for neurodegenerative diseases are essential for implementing large-scale clinical studies. A reliable and validated blood test should require a small sample volume as well as be a less invasive sampling method, affordable, and reproducible. This paper demonstrates that high-throughput capillary electrophoresis immunoassay satisfies criteria for potential biomarker development.

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Developmental Biology

Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes
Jeremy P. Goering *1, Dona Greta Isai *1, Andras Czirok 1,2, Irfan Saadi 1
1Department of Anatomy and Cell Biology, University of Kansas Medical Center, 2Department of Biological Physics, Eotvos University

We present a protocol for isolation and culture of primary mouse embryonic palatal mesenchymal cells for time-lapse imaging of two-dimensional (2D) growth and wound-repair assays. We also provide the methodology for analysis of the time-lapse imaging data to determine cell-stream formation and directional motility.

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Medicine

Software-Assisted Quantitative Measurement of Osteoarthritic Subchondral Bone Thickness
Xiangliang Liu *1, Michael A. Pitner *1, Patrick P. Baki 1, Qinghua Lu 1, John P. Schroeppel 2, Jinxi Wang 1,3
1Harrington Laboratory for Molecular Orthopedics, Department of Orthopedic Surgery, University of Kansas Medical Center, 2Division of Sports Medicine, Department of Orthopedic Surgery, University of Kansas Medical Center, 3Department of Biochemistry & Molecular Biology, University of Kansas Medical Center

This methodology article presents a software-assisted quantitative measurement protocol to quantify histologic subchondral bone thickness in murine osteoarthritic knee joints and normal knee joints as controls. This protocol is highly sensitive to subtle thickening and is suitable for detecting early osteoarthritic subchondral bone changes.

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Biology

Single-Particle Interferometric Reflectance Imaging Characterization of Individual Extracellular Vesicles and Population Dynamics
Fengyan Deng 1, Anamika Ratri 1, Clayton Deighan 2, George Daaboul 2, Paige C. Geiger 1, Lane K. Christenson 1
1Department of Molecular and Integrative Physiology, University of Kansas Medical Center, 2NanoView Biosciences

This protocol presents single-particle interferometric reflectance imaging that is designed for the multi-level and comprehensive measurements of extracellular vesicles (EV) size, EV count, EV phenotype, and EV biomarker colocalization.

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Bioengineering

Improving Reproducibility to Meet Minimal Information for Studies of Extracellular Vesicles 2018 Guidelines in Nanoparticle Tracking Analysis
Orman L. Snyder 1, Alexander W. Campbell 2, Lane K. Christenson 2, Mark L. Weiss 1
1Department of Anatomy and Physiology, Kansas State University College of Veterinary Medicine, 2Department of Molecular and Integrative Physiology, University of Kansas Medical Center

Nanoparticle tracking analysis (NTA) is a widely used method to characterize extracellular vesicles. This paper highlights NTA experimental parameters and controls plus a uniform method of analysis and characterization of samples and diluents necessary to supplement the guidelines proposed by MISEV2018 and EV-TRACK for reproducibility between laboratories.

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Cancer Research

Rapid In Vitro Cytotoxicity Evaluation of Jurkat Expressing Chimeric Antigen Receptor using Fluorescent Imaging
Siddharth Subham *1,2,3, John D. Jeppson *1, Benjamin K. Gibbs 4, Jacqueline Babai 2, Riza Alker 1, Andrew K. Godwin 4,5,6, David Akhavan 1,2,3
1Department of Radiation Oncology, University of Kansas Cancer Center, 2Department of Cancer Biology, University of Kansas Cancer Center, 3BioEngineering Program, University of Kansas, 4Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, 5University of Kansas Cancer Center, 6Kansas Institute for Precision Medicine, University of Kansas Medical Center

A protocol to evaluate quantitative tumor cell killing by Jurkat cells expressing chimeric antigen receptor (CAR) targeting single tumor antigen. This protocol can be used as a screening platform for rapid optimization of CAR hinge constructs prior to confirmation in peripheral blood-derived T cells.

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Medicine

Multi-modal Pulmonary Imaging: Using Complementary Information from CT and Hyperpolarized 129Xe MRI to Evaluate Lung Structure-Function
Rachel L Eddy 1,2, George H Xu 3, Jonathon A Leipsic 1,4, Janice M Leung 1,2, Don D Sin 1,2, Chase S Hall 5, Roger C Tam 3,4
1UBC Centre for Heart Lung Innovation, St. Paul’s Hospital, 2Division of Respiratory Medicine, Department of Medicine, University of British Columbia, 3School of Biomedical Engineering, University of British Columbia, 4Department of Radiology, University of British Columbia, 5Division of Pulmonary and Critical Care Medicine, University of Kansas Medical Center

CT and 129Xe MRI provide complementary lung structure-function information that can be exploited for regional analysis using image registration. Here, we provide a protocol that builds from the existing literature for 129Xe MR to CT image registration using open-source platforms.

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Bioengineering

Hyperpolarized 129Xe Lung MRI and Spectroscopy in Mechanically Ventilated Mice
Mariah L. Costa 1,2,3, Joseph W. Plummer 1,2, Abdullah S. Bdaiwi 1, Brice J. Albert 4, Elizabeth M. Fugate 3, Peter J. Niedbalski 5,6,7, Diana M. Lindquist 3,8, Zackary I. Cleveland 1,2,3,8,9
1Center for Pulmonary Imaging Research, Division of Pulmonary Medicine, Cincinnati Children's Hospital Medical Center, 2Department of Biomedical Engineering, University of Cincinnati, 3Imaging Research Center, Department of Radiology, Cincinnati Children's Hospital Medical Center, 4Analytical Technology Group, Aurorium, 5Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Internal Medicine, University of Kansas Medical Center, 6Department of Bioengineering, University of Kansas, 7Hoglund Biomedical Imaging Center, University of Kansas Medical Center, 8Department of Pediatrics, University of Cincinnati, 9Division of Pulmonary Medicine, Cincinnati Children's Hospital Medical Center

Hyperpolarized xenon MRI can quantify regional lung microstructure (air-space dimensions) and physiology (ventilation and gas exchange) in translational research and clinical care. Although challenging, it can provide comparable pulmonary insights in preclinical studies. This protocol describes the infrastructure and procedures needed to perform routine xenon lung MRI in mice.

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Medicine

Pulmonary Structural MRI using Free-Breathing, Self-Gated Ultra-short Echo Time Imaging
Peter J. Niedbalski 1
1Division of Pulmonary, Critical Care, and Sleep Medicine, Hoglund Biomedical Imaging Center, University of Kansas Medical Center

A protocol is described for generating high-resolution structural images of the lungs using ultra-short-echo time (UTE) Magnetic Resonance Imaging (MRI). This protocol allows for images to be acquired using a simple MRI pulse sequence during free-breathing.

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