University of Kansas Medical Center 17 articles published in JoVE 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. Gibbs4, Jacqueline Babai2, Riza Alker1, Andrew K. Godwin4,5,6, David Akhavan1,2,3 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. Medicine Software-Assisted Quantitative Measurement of Osteoarthritic Subchondral Bone Thickness Xiangliang Liu*1, Michael A. Pitner*1, Patrick P. Baki1, Qinghua Lu1, John P. Schroeppel2, Jinxi Wang1,3 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. Bioengineering Improving Reproducibility to Meet Minimal Information for Studies of Extracellular Vesicles 2018 Guidelines in Nanoparticle Tracking Analysis Orman L. Snyder1, Alexander W. Campbell2, Lane K. Christenson2, Mark L. Weiss1 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. 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 Czirok1,2, Irfan Saadi1 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. Medicine Imaging Features of Systemic Sclerosis-Associated Interstitial Lung Disease Jonathan H. Chung1, Christopher M. Walker2, Stephen Hobbs3 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. Neuroscience Use of Capillary Electrophoresis Immunoassay to Search for Potential Biomarkers of Amyotrophic Lateral Sclerosis in Human Platelets Jessica M. Sage1, LaSharice Hall2, April McVey3, Richard J. Barohn3, Jeffrey M. Statland3, Omar Jawdat3, Mazen M. Dimachkie3, Abdulbaki Agbas1 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. Neuroscience Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain Prabhakar Singh1, David W. Peng1, William Z. Suo1,2,3,4 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, 4 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. Biology Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry Shriram Venkatesan*1, Tejbir S. Kandola*1, Alejandro Rodríguez-Gama1, Andrew Box1, Randal Halfmann1,2 1Stowers Institute for Medical Research, 2Department of Molecular and Integrative Physiology, The University of Kansas School of Medicine This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells. Biology Gamete Collection and In Vitro Fertilization of Astyanax mexicanus Robert Peuß*1, Zachary Zakibe*1, Jaya Krishnan1, M. Shane Merryman1, Diana P. Baumann1, Nicolas Rohner1,2 1Stowers Institute for Medical Research, 2Department of Molecular & Integrative Physiology, KU Medical Center In vitro fertilization is a commonly used technique with a variety of model organisms to maintain lab populations and produce synchronized embryos for downstream applications. Here, we present a protocol that implements this technique for different populations of the Mexican tetra fish, Astyanax mexicanus. 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 Seng1, Mary A. Markiewicz1 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. Genetics Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events Melvin Noe Gonzalez1, Joan W. Conaway1,2, Ronald C. Conaway1,2 1Stowers Institute for Medical Research, 2Department of Biochemistry and Molecular Biology, Kansas University Medical Center Here, we describe the assembly of RNA polymerase II (Pol II) elongation complexes requiring only short synthetic DNA and RNA oligonucleotides and purified Pol II. These complexes are useful for studying mechanisms underlying co-transcriptional processing of transcripts associated with the Pol II elongation complex. Biochemistry Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy Alexandra J. Machen1, Pierce T. O'Neil1, Bradley L. Pentelute2, Maria T. Villar1, Antonio Artigues1, Mark T. Fisher1 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. Bioengineering A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering Christopher A. Neal1, Jennifer G. Nelson-Brantley1, Michael S. Detamore2, Hinrich Staecker1, Adam J. Mellott3 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. Neuroscience Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction Mychal S. Grames1, Kasey L. Jackson1, Robert D. Dayton1, John A. Stanford2, Ronald L. Klein1 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. Medicine Assessment of Perigenital Sensitivity and Prostatic Mast Cell Activation in a Mouse Model of Neonatal Maternal Separation Isabella M. Fuentes1, Angela N. Pierce1, Pierce T. O'Neil1, Julie A. Christianson1 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. Biology Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes Lu Chen1,2, Soon-Keat Ooi1, Joan W. Conaway1,2, Ronald C. Conaway1,2 1Stowers Institute for Medical Research, 2Department of Biochemistry & Molecular Biology, Kansas University Medical Center Here we describe biochemical assays that can be used to characterize ATP-dependent chromatin remodeling enzymes for their abilities to 1) catalyze ATP-dependent nucleosome sliding, 2) engage with nucleosome substrates, and 3) hydrolyze ATP in a nucleosome- or DNA-dependent manner. Biology Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes Lu Chen1,2, Soon-Keat Ooi1, Ronald C. Conaway1,2, Joan W. Conaway1,2 1Stowers Institute for Medical Research, 2Department of Biochemistry & Molecular Biology, Kansas University Medical Center This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.
