Mayo Clinic View Institution's Website 47 articles published in JoVE Cancer Research Positron Emission Tomography Imaging of Cell Trafficking: A Method of Cell Radiolabeling Aditya Bansal1, Timothy R. DeGrado2, Mukesh K. Pandey1 1Division of Nuclear Medicine, Department of Radiology, Mayo Clinic, 2Department of Radiology, University of Colorado Anschutz Medical Campus Presented here is a protocol to radiolabel cells with a positron emission tomography (PET) radioisotope, 89Zr (t1/2 78.4 h), using a ready-to-use radiolabeling synthon, [89Zr]Zr-p-isothiocyanatobenzyl-desferrioxamine ([89Zr]Zr-DBN). Radiolabeling cells with [89Zr]Zr-DBN allows noninvasive tracking and imaging of administered radiolabeled cells in the body with PET for up to 7 days post-administration. Cancer Research Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-Derived Xenograft Mouse Model Claudia Manriquez Roman1,2,3,4,5, R. Leo Sakemura1,2, Brooke L. Kimball1,2, Fang Jin6, Roman H. Khadka6, Mohamad M. Adada1,2, Elizabeth L. Siegler1,2, Aaron J. Johnson6, Saad S. Kenderian1,2,6 1T Cell Engineering Laboratory, Mayo Clinic, Rochester, 2Division of Hematology, Mayo Clinic, Rochester, 3Mayo Clinic Graduate School of Biomedical Sciences, Mayo Clinic, Rochester, 4Department of Molecular Medicine, Mayo Clinic, Rochester, 5Regenerative Sciences PhD Program, Mayo Clinic, Rochester, 6Department of Immunology, Mayo Clinic, Rochester Here, we describe a protocol in which an acute lymphoblastic leukemia patient-derived xenograft model is used as a strategy to assess and monitor CD19-targeted chimeric antigen receptor T cell-associated toxicities. Medicine Studying Murine Small Bowel Mechanosensing of Luminal Particulates Arnaldo Mercado-Perez*1,2, Andrew Wegner*1, Kaitlyn Knutson1, Michael Zumchak3, Arthur Beyder1,4 1Enteric NeuroScience Program (ENSP), Division of Gastroenterology & Hepatology, Department of Medicine, Mayo Clinic, 2Medical Scientist Training Program (MSTP), Mayo Clinic, 3Easy Learning Labs, 4Department of Physiology & Biomedical Engineering (PBME), Mayo Clinic To study how the small bowel handles particulates of varying sizes, we have modified an established in vivo method to determine small bowel transit. Cancer Research Dynamic Imaging of Chimeric Antigen Receptor T Cells with [18F]Tetrafluoroborate Positron Emission Tomography/Computed Tomography Reona Sakemura1,2, Michelle J. Cox1,2, Aditya Bansal3, Claudia Manriquez Roman1,2,4,5, Mehrdad Hefazi1,2, Cynthia J. Vernon3, Dianna L. Glynn3, Mukesh K. Pandey3, Timothy R. DeGrado3, Elizabeth L. Siegler1,2, Saad S. Kenderian1,2,5,6 1T Cell Engineering, Mayo Clinic, 2Division of Hematology, Mayo Clinic, 3Department of Radiology, Mayo Clinic, 4Regenerative Sciences PhD, Mayo Clinic, 5Department of Molecular Medicine, Mayo Clinic, 6Department of Immunology, Mayo Clinic This protocol describes the methodology for non-invasively tracking T cells genetically engineered to express chimeric antigen receptors in vivo with a clinically available platform. Bioengineering Characterization of Blood Outgrowth Endothelial Cells (BOEC) from Porcine Peripheral Blood Akankshya Shradhanjali1, Susheil Uthamaraj2, Dan Dragomir-Daescu3, Rajiv Gulati4, Gurpreet S. Sandhu4, Brandon J. Tefft1 1Department of Biomedical Engineering, Medical College of Wisconsin & Marquette University, 2Division of Engineering, Mayo Clinic, 3Department of Physiology & Biomedical Engineering, Mayo Clinic, 4Department of Cardiovascular Medicine, Mayo Clinic Biology Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells Qianqian Guo1, Kunimaro Furuta1, Ahmed Aly1, Samar H. Ibrahim1,2 1Division of Gastroenterology & Hepatology, Mayo Clinic, 2Division of Pediatric Gastroenterology, Mayo Clinic Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy. Bioengineering Microfluidic Fabrication of Core-Shell Microcapsules carrying Human Pluripotent Stem Cell Spheroids Kihak Gwon*1, Hye Jin Hong*1, Alan M. Gonzalez-Suarez1, Gulnaz Stybayeva1, Alexander Revzin1 1Department of Physiology and Biomedical Engineering, Mayo Clinic This article describes encapsulation of human pluripotent stem cells (hPSCs) using a co-axial flow focusing device. We demonstrate that this microfluidic encapsulation technology enables efficient formation of hPSC spheroids. Medicine Use of 3D Robotic Ultrasound for In Vivo Analysis of Mouse Kidneys Heather L. Holmes*1, Alison A. Stiller*1, Christopher J. Moore4, Adriana V. Gregory2, Carolyn M. Roos3, Jordan D. Miller1,3, Ryan C. Gessner4, Tomasz J. Czernuszewicz4, Timothy L. Kline2, Michael F. Romero1,5 1Physiology & Biomedical Engineering, Mayo Clinic, 2Radiology, Mayo Clinic, 3Surgery, Mayo Clinic, 4SonoVol, Inc., 5Nephrology & Hypertension, Mayo Clinic This protocol demonstrates robotic ultrasound (US) as a practical, cost-effective, and quick alternative to traditional non-invasive image modalities. Neuroscience Maximum Isometric Tetanic Force Measurement of the Tibialis Anterior Muscle in the Rat Meiwand Bedar1,2, Tiam M. Saffari1,2, Patricia F. Friedrich1, Guilherme Giusti1, Allen T. Bishop1, Alexander Y. Shin1 1Department of Orthopedic Surgery, Mayo Clinic, 2Radboud University Medical Center, Radboud Institute for Health Sciences, Department of Plastic Surgery, Nijmegen, The Netherlands Evaluation of motor recovery remains the benchmark outcome measure in experimental peripheral nerve studies. The isometric tetanic force measurement of the tibialis anterior muscle in the rat is an invaluable tool to assess functional outcomes after reconstruction of sciatic nerve defects. The methods and nuances are detailed in this article. Behavior A Complex Diving-For-Food Task to Investigate Social Organization and Interactions in Rats Benjamin F. Gruenbaum*1, Dmitry Frank*2, Shiri Savir2, Honore N. Shiyntum3, Ruslan Kuts2, Max Vinokur2, Israel Melamed4, Michael Dubilet2, Alexander Zlotnik2, Matthew Boyko2 1Department of Anesthesiology and Perioperative Medicine, Mayo Clinic, 2Division of Anesthesiology and Critical Care, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev, 3 This protocol describes a method of examining social hierarchy in a rat model. Rats perform a complex diving-for-food task in which they form a distinct hierarchy according to their willingness to dive underwater and swim to obtain a food pellet. This method is used to understand decision making and social relationships among highly social animals in small groups. Medicine Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model Michaela Olthoff1, Federico Franchi1, Karen M. Peterson1, Ramasamy Paulmurugan2, Martin Rodriguez-Porcel1 1Department of Cardiovascular Medicine, Mayo Clinic, 2Department of Radiology and Molecular Imaging Program at Stanford (MIPS), Stanford University School of Medicine Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury. Biology Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues Dong Seong Cho1, Jason D. Doles1 1Department of Biochemistry and Molecular Biology, Mayo Clinic We present a simple method to isolate highly viable adipose progenitor cells from mouse epididymal fat pads using fluorescence activated cell sorting. Cancer Research Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts Piyan Zhang1, Irina V. Kovtun1,2 1Center for Individualized Medicine, Mayo Clinic, 2Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients’ tumors into mice and testing responses to corresponding drugs. Biochemistry Isolation of Tissue Extracellular Vesicles from the Liver Kaori Ishiguro1, Irene K. Yan1, Tushar Patel1 1Department of Transplantation, Mayo Clinic This is a protocol to isolate tissue extracellular vesicles (EVs) from the liver. The protocol describes a two-step process involving collagenase perfusion followed by differential ultracentrifugation to isolate liver tissue EVs. Cancer Research Using CRISPR/Cas9 to Knock Out GM-CSF in CAR-T Cells Rosalie M. Sterner1,2, Michelle J. Cox3, Reona Sakemura3, Saad S. Kenderian2,3 1Mayo Clinic Medical Scientist Training Program, Mayo Clinic College of Medicine and Science, 2Department of Immunology, Mayo Clinic, 3Division of Hematology, Mayo Clinic Here, we present a protocol to genetically edit CAR-T cells via a CRISPR/Cas9 system. Medicine Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine Robert A. Kaiser1,2, Shennen A. Mao1, Jaime Glorioso3, Bruce Amiot1, Clara T. Nicolas1, Kari L. Allen1, Zeji Du1, Caitlin J VanLith1, Raymond D. Hickey1, Scott L. Nyberg1, Joseph B. Lillegard1,2,4 1Department of Surgery, Mayo Clinic, 2 This protocol is intended to describe porcine hepatocyte isolation and ex vivo gene delivery to cure models of metabolic diseases via autologous cell transplantation. Although this particular model enjoys unique advantages that favor successful therapy, the application is a relevant foundation to address additional diseases and indications. Medicine Conducting Maximal and Submaximal Endurance Exercise Testing to Measure Physiological and Biological Responses to Acute Exercise in Humans Courtney M. Wheatley1, Triven Kannan1, Svetlana Bornschlegl2, Chul-ho Kim1, Dennis A. Gastineau2, Allan B. Dietz2, Bruce D. Johnson1, Michael P. Gustafson2 1Department of Cardiovascular Diseases, Mayo Clinic, 2Department of Laboratory Medicine and Pathology, Mayo Clinic To assess the influence of exercise intensity on physiologic and biologic responses, two different exercise testing protocols were utilized. Methods outlining exercise testing on a cycle ergometer as an incremental maximal oxygen consumption test and endurance, steady state submaximal endurance test are described. Biology Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells Katilyn Knutson*1, Peter R. Strege*1, Joyce Li2, Andrew B. Leiter2, Gianrico Farrugia1, Arthur Beyder1 1Enteric Neuroscience Program, Division of Gastroenterology & Hepatology,Department of Physiology & Biomedical Engineering, Mayo Clinic, 2Division of Gastroenterology, Department of Medicine, University of Massachusetts Medical School Enterochromaffin (EC) cells comprise a small subset of gastrointestinal epithelial cells. EC cells are electrically excitable and release serotonin, yet difficulties in culturing and identifying EC cells have limited physiological studies. The method presented here establishes a primary culture model amenable to examination of single EC cells by electrophysiology. Medicine A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish Xiao Ma*1,2,3, Yonghe Ding*2,3, Yong Wang2,3,4, Xiaolei Xu1,2,3 1Clinical and Translational Sciences Track, Mayo Clinic Graduate School of Biomedical Sciences, 2Department of Biochemistry and Molecular Biology, Mayo Clinic, 3Division of Cardiovascular Diseases, Mayo Clinic, 4Institute of Life Science, Beijing University of Chinese Medicine A method to generate a doxorubicin-induced cardiomyopathy model in adult zebrafish (Danio rerio) is described here. Two alternative ways of intraperitoneal injection are presented and conditions to reduce variations among different experimental groups are discussed. Biology Endotracheal Intubation of Rabbits Using a Polypropylene Guide Catheter Krista L Thompson1, Thomas R Meier1, Jodi A Scholz1 1Department of Comparative Medicine, Mayo Clinic Endotracheal intubation in rabbits is challenging due to their unusual anatomy. Here we present a technique for direct intubation of the trachea using a polypropylene catheter as a guide. This method utilizes relatively inexpensive supplies, requires minimal training, and can be easily performed in any clinical setting. Neuroscience Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine Mark D. Unger1, Timothy P. Maus2, Ross C. Puffer3, Laura K. Newman1, Bradford L. Currier4, Andreas S. Beutler1 1Departments of Anesthesiology and Oncology, Mayo Clinic, Translational Science Track, Mayo Graduate School, 2Department of Radiology (Section of Interventional Pain Management), Mayo Clinic, 3Department of Neurologic Surgery, Mayo Clinic, 4Department of Orthopedic Surgery, Mayo Clinic We describe a method for laminotomy in swine that provides access to lumbar dorsal root ganglia (DRG) for intraganglionic injection. Injection progress is monitored intraoperatively and histologically confirmed up to 21 days after surgery. This protocol could be used for future preclinical studies involving DRG injection. Bioengineering A Method to Estimate Cadaveric Femur Cortical Strains During Fracture Testing Using Digital Image Correlation Timothy Rossman1, Susheil Uthamaraj1, Asghar Rezaei1,2, Sean McEligot1, Hugo Giambini3, Iwona Jasiuk4, Michael J. Yaszemski3, Lichun Lu3, Dan Dragomir-Daescu1,2 1Division of Engineering, Mayo Clinic, 2Department of Physiology and Biomedical Engineering, Mayo Clinic, 3Department of Orthopedic Surgery, Mayo Clinic, 4Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign In this protocol, the femur surface strains are estimated during fracture testing using the digital image correlation technique. The novelty of the method involves application of a high-contrast stochastic speckle pattern on the femur surface, carefully specified illumination, high speed video capture, and digital image correlation analysis for strain calculations. Bioengineering Method and Instrumented Fixture for Femoral Fracture Testing in a Sideways Fall-on-the-Hip Position Dan Dragomir-Daescu1,2, Asghar Rezaei1,2, Timothy Rossman2, Susheil Uthamaraj2, Rachel Entwistle2, Sean McEligot2, Vincent Lambert2, Hugo Giambini3, Iwona Jasiuk4, Michael J. Yaszemski3, Lichun Lu1,3 1Department of Physiology and Biomedical Engineering, Mayo Clinic, 2Division of Engineering, Mayo Clinic, 3Department of Orthopedic Surgery, Mayo Clinic, 4Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign In this manuscript, we present a protocol to fracture test cadaveric proximal femora in a sideways fall on the hip configuration using instrumented fixtures mounted on a standard servo hydraulic frame. Nine digitized signals comprising forces, moments, and displacement along with two high speed video streams are acquired during testing. Medicine Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model Dimitra Kotsougiani1, Caroline A. Hundepool1, Joost I. Willems1, Patricia Friedrich1, Alexander Y. Shin1, Allen T. Bishop1 1Microvascular Research Laboratory, Department of Orthopedic Surgery, Mayo Clinic Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation. Biology Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator Adam J. Rossano1, Michael F. Romero1,2 1Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, 2Department of Nephrology and Hypertension, Mayo Clinic College of Medicine Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator. Medicine Proximal Cadaveric Femur Preparation for Fracture Strength Testing and Quantitative CT-based Finite Element Analysis Dan Dragomir-Daescu1,2, Asghar Rezaei1,2, Susheil Uthamaraj2, Timothy Rossman2, James T. Bronk3, Mark Bolander3, Vincent Lambert2, Sean McEligot2, Rachel Entwistle2, Hugo Giambini3, Iwona Jasiuk4, Michael J. Yaszemski3, Lichun Lu1,3 1Department of Physiology and Biomedical Engineering, Mayo Clinic, 2Division of Engineering, Mayo Clinic, 3Department of Orthopedic Surgery, Mayo Clinic, 4Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign We present a robust protocol on how to carefully preserve and prepare cadaveric femora for fracture testing and quantitative computed tomography imaging. The method provides precise control over input conditions for the purpose of determining relationships between bone mineral density, fracture strength, and defining finite element model geometry and properties. Developmental Biology Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy Manuela Lahne1, Ryne A. Gorsuch1, Craig M. Nelson1,2, David R. Hyde1 1Department of Biological Sciences, University of Notre Dame, 2Department of Neurosurgery, Mayo Clinic Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy. Medicine Echocardiographic Approaches and Protocols for Comprehensive Phenotypic Characterization of Valvular Heart Disease in Mice Grace Casaclang-Verzosa1, Maurice Enriquez-Sarano2, Hector R. Villaraga2, Jordan D. Miller1,3 1Department of Surgery, Mayo Clinic, 2Department of Medicine, Mayo Clinic, 3Department of Physiology & Biomedical Engineering, Mayo Clinic This protocol provides a detailed description of the echocardiographic approach for comprehensive phenotyping of heart and heart valve function in mice. Immunology and Infection MicroRNA-based Regulation of Picornavirus Tropism Autumn J. Ruiz1, Stephen J. Russell1 1Department of Molecular Medicine, Mayo Clinic College of Medicine We describe here a method for regulating picornavirus tropism by incorporating sequences complementary to specific microRNAs into the viral genome. This protocol can be adapted to all different classes of viruses with modifications based upon the length and nature of their life cycle. Medicine Fabrication of Small Caliber Stent-grafts Using Electrospinning and Balloon Expandable Bare Metal Stents Susheil Uthamaraj1, Brandon J. Tefft2, Soumen Jana2, Ota Hlinomaz3, Manju Kalra4, Amir Lerman2, Dan Dragomir-Daescu1,5, Gurpreet S. Sandhu2 1Division of Engineering, Mayo Clinic, 2Department of Cardiovascular Diseases, Mayo Clinic, 3 In the protocol, we present a method to manufacture a small caliber stent-graft by sandwiching a balloon expandable stent between two electrospun nanofibrous polyurethane layers. Medicine Intravascular Delivery of Biologics to the Rat Kidney Federico Franchi1, Xiang Yang Zhu2, Tyra A. Witt1, Lilach O. Lerman2, Martin Rodriguez-Porcel1 1Divisions of Cardiovascular Diseases, Department of Internal Medicine, Mayo Clinic, 2Divisions of Nephrology and Hypertension, Department of Internal Medicine, Mayo Clinic The administration of drugs for recovery of kidney function requires control of the localization and distribution of the therapeutic compound. Here, we describe in detail a simple technique for intrarenal delivery of drugs in rats. This procedure may be easily performed with no mortality and high reproducibility. Immunology and Infection Antibody Binding Specificity for Kappa (Vκ) Light Chain-containing Human (IgM) Antibodies: Polysialic Acid (PSA) Attached to NCAM as a Case Study Jens O. Watzlawik1,2,3, Robert J. Kahoud1,3,5, Bharath Wootla1,2,3, Meghan M. Painter1,2,3, Arthur E. Warrington1,2,3, William A. Carey3,4, Moses Rodriguez1,2,3 1Department of Neurology, Mayo Clinic, 2Mayo Clinic Center for Multiple Sclerosis and Autoimmune Neurology, Mayo Clinic, 3Center for Regenerative Medicine, Neuroregeneration, Mayo Clinic, 4Division of Neonatal Medicine, Mayo Clinic, 5Department of Pediatric and Adolescent Medicine, Mayo Clinic We present a protocol to identify protein moieties containing antigens for human and mouse IgM antibodies with specific kappa (Vκ) light chains. This protocol is not limited to IgM class antibodies but applies to all immunoglobulin isotypes that target their antigen with sufficiently high affinity during immunoprecipitations. Bioengineering Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles Brandon J. Tefft1, Susheil Uthamaraj2, J. Jonathan Harburn3, Martin Klabusay4, Dan Dragomir-Daescu2,5, Gurpreet S. Sandhu1 1Division of Cardiovascular Diseases, Mayo Clinic, 2Division of Engineering, Mayo Clinic, 3School of Medicine, Pharmacy and Health, Durham University, 4Regional Center for Applied Molecular Oncology, Masaryk Memorial Cancer Institute, 5Mayo Clinic College of Medicine, Mayo Clinic Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization. Bioengineering Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention Susheil Uthamaraj1, Brandon J. Tefft2, Ota Hlinomaz3, Gurpreet S. Sandhu2, Dan Dragomir-Daescu1,4 1Division of Engineering, Mayo Clinic, 2Division of Cardiovascular Diseases, Mayo Clinic, 3 Our goals were to design, manufacture and test ferromagnetic stents for endothelial cell capture. Ten stents were tested for fracture and 10 more stents were tested for retained magnetism. Finally, 10 stents were tested in-vitro and 8 more stents were implanted in 4 pigs to show cell capture and retention. Medicine Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring Maria V. Irazabal1, Prasanna K. Mishra2, Vicente E. Torres1, Slobodan I. Macura2 1Department of Internal Medicine, Division of Nephrology, Mayo Clinic, 2Department of Biochemistry and Molecular Biology, Mayo Clinic The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up. Medicine Reduction of Iatrogenic Atrial Septal Defects with an Anterior and Inferior Transseptal Puncture Site when Operating the Cryoballoon Ablation Catheter Michael E. Rich1, Andrew Tseng2, Hae W. Lim3, Paul J. Wang4, Wilber W. Su1 1Department of Cardiovascular Medicine, Cavanagh Heart Center, Banner-University Medical Center, 2Mayo Medical School, Mayo Clinic, 3AF Solutions, Medtronic plc, 4Cardiology Division, Stanford University School of Medicine, Stanford University The goal of this study is to demonstrate the preferential location of transseptal puncture during a cryoballoon catheter ablation procedure for the treatment of atrial fibrillation. Medicine Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions Sofia Karkampouna1, Peter Kloen2, Miryam C. Obdeijn3, Scott M. Riester4, Andre J. van Wijnen4,5, Marianna Kruithof-de Julio1,6 1Department of Molecular Cell Biology, Cancer Genomics Centre and Centre for Biomedical Genetics, Leiden University Medical Center, 2Department of Orthopedic Surgery, Academic Medical Center, 3Department of Plastic, Reconstructive and Hand Surgery, Academic Medical Center, 4Department of Orthopedic Surgery, Mayo Clinic, 5Department of Biochemistry and Molecular Biology, Mayo Clinic, 6Department of Dermatology, Leiden University Medical Center Dupuytren’s disease (DD) is a fibroproliferative disease of the palm of the hand. Here, we present a protocol to culture resection specimens from DD in a three-dimensional (3D) culture system. Such short-term culture system allows preservation of the 3D structure and molecular properties of the fibrotic tissue. Developmental Biology Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis Choong Yong Ung1, Feng Guo2, Xiaoling Zhang3, Zhihui Zhu3, Shizhen Zhu1,3 1Department of Molecular Pharmacology & Experimental Therapeutics, Mayo Clinic College of Medicine, Center for Individualized Medicine, 2Tufts University School of Medicine, 3Department of Biochemistry and Molecular Biology, Mayo Clinic The goal of this study is to demonstrate how the mosaic transgenesis strategy can be used in zebrafish to rapidly and efficiently assess the relative contributions of multiple oncogenes in tumor initiation and progression in vivo. Bioengineering Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method Nathan J. Wittenberg1, Timothy W. Johnson1, Luke R. Jordan2, Xiaohua Xu3, Arthur E. Warrington3, Moses Rodriguez3,4, Sang-Hyun Oh1,2 1Department of Electrical and Computer Engineering, University of Minnesota, 2Department of Biomedical Engineering, University of Minnesota, 3Department of Neurology, Mayo Clinic College of Medicine, 4Department of Immunology, Mayo Clinic College of Medicine Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles. Neuroscience Heat-Induced Antigen Retrieval: An Effective Method to Detect and Identify Progenitor Cell Types during Adult Hippocampal Neurogenesis Syed M.Q. Hussaini*1, Heechul Jun*1, Chang Hoon Cho1, Hyo Jin Kim1, Woon Ryoung Kim2, Mi-Hyeon Jang1,3 1Department of Neurologic Surgery, Mayo Clinic College of Medicine, 2Department of Anatomy, Korea University College of Medicine, 3Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine During adult hippocampal neurogenesis, a distinct set of genetic markers are expressed when a quiescent neural stem cell sequentially progresses and develops into a functionally integrated neuron in the circuit. Using heat-induced antigen retrieval, progenitor cell types that are otherwise difficult to detect are identified with improved effectiveness. Medicine Assessment of Gastric Emptying in Non-obese Diabetic Mice Using a [13C]-octanoic Acid Breath Test Christopher T. Creedon1, Pieter-Jan Verhulst1, Kyoung M. Choi1, Jessica E. Mason1, David R. Linden1, Joseph H. Szurszewski1, Simon J. Gibbons1, Gianrico Farrugia1 1Enteric Neuroscience Program, Department of Physiology and Biomedical Engineering, Mayo Clinic Determination of gastric emptying with a non-invasive [13C]-octanoic acid breath test for tracking gastroparesis in female NOD LtJ mice. Immunology and Infection Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System Courtney L. Erskine1, Andrea M. Henle1, Keith L. Knutson1 1Department of Immunology, College of Medicine, Mayo Clinic The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing. Biology Immunostaining of Dissected Zebrafish Embryonic Heart Jingchun Yang1, Xiaolei Xu1 1Division of Cardiovascular Diseases, Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time. Immunology and Infection Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format Leslie Hall1, Kurt P. Jude1, Shirley L. Clark1, Nancy L. Wengenack1 1Department of Laboratory Medicine and Pathology, Mayo Clinic Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment. Immunology and Infection Isolation of Brain-infiltrating Leukocytes Reghann G. LaFrance-Corey*1, Charles L. Howe*1 1Department of Neurology, Mayo Clinic College of Medicine A rapid method to obtain infiltrating leukocytes from the murine brain is described. This method utilizes a continuous Percoll gradient and discontinuous Ficoll gradient to select and purify the leukocyte-enriched layer. Isolated leukocytes may then be characterized by flow cytometric measurements. Biology IP-FCM: Immunoprecipitation Detected by Flow Cytometry Tessa R. Davis1, Adam G. Schrum1 1Department of Immunology, College of Medicine, Mayo Clinic The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes. Biology In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer Alexis B. Cordero1, Youngjoo Kwon1, Xiang Hua2, Andrew K. Godwin1 1Department of Medical Oncology, Women's Cancer Program, 2Transgenic Mouse Facility, Fox Chase Cancer Center Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.