Mayo Clinic

44 articles published in JoVE

• Medicine

  
  Studying Murine Small Bowel Mechanosensing of Luminal Particulates Arnaldo Mercado-Perez*^1,2, Andrew Wegner*¹, Kaitlyn Knutson¹, Michael Zumchak³, Arthur Beyder^{1,4 1}Enteric 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 Sakemura^1,2, Michelle J. Cox^1,2, Aditya Bansal³, Claudia Manriquez Roman^1,2,4,5, Mehrdad Hefazi^1,2, Cynthia J. Vernon³, Dianna L. Glynn³, Mukesh K. Pandey³, Timothy R. DeGrado³, Elizabeth L. Siegler^1,2, Saad S. Kenderian^{1,2,5,6 1}T 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.

• Biology

  
  Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells Qianqian Guo¹, Kunimaro Furuta¹, Ahmed Aly¹, Samar H. Ibrahim^{1,2 1}Division 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 Revzin^{1 1}Department 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. Miller^1,3, Ryan C. Gessner⁴, Tomasz J. Czernuszewicz⁴, Timothy L. Kline², Michael F. Romero^{1,5 1}Physiology & 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 Bedar^1,2, Tiam M. Saffari^1,2, Patricia F. Friedrich¹, Guilherme Giusti¹, Allen T. Bishop¹, Alexander Y. Shin^{1 1}Department 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 Boyko^{2 1}Department 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-Porcel^{1 1}Department 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. Doles^{1 1}Department 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. Kovtun^{1,2 1}Center 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 Patel^{1 1}Department 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. Sterner^1,2, Michelle J. Cox³, Reona Sakemura³, Saad S. Kenderian^{2,3 1}Mayo 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. Kaiser^1,2, 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. Lillegard^{1,2,4 1}Department 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. Gustafson^{2 1}Department 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 Beyder^{1 1}Enteric 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 Wang^2,3,4, Xiaolei Xu^{1,2,3 1}Clinical 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 Scholz^{1 1}Department 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. Beutler^{1 1}Departments 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 Rezaei^1,2, Sean McEligot¹, Hugo Giambini³, Iwona Jasiuk⁴, Michael J. Yaszemski³, Lichun Lu³, Dan Dragomir-Daescu^{1,2 1}Division 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-Daescu^1,2, Asghar Rezaei^1,2, Timothy Rossman², Susheil Uthamaraj², Rachel Entwistle², Sean McEligot², Vincent Lambert², Hugo Giambini³, Iwona Jasiuk⁴, Michael J. Yaszemski³, Lichun Lu^{1,3 1}Department 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. Bishop^{1 1}Microvascular 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. Romero^{1,2 1}Department 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-Daescu^1,2, Asghar Rezaei^1,2, Susheil Uthamaraj², Timothy Rossman², James T. Bronk³, Mark Bolander³, Vincent Lambert², Sean McEligot², Rachel Entwistle², Hugo Giambini³, Iwona Jasiuk⁴, Michael J. Yaszemski³, Lichun Lu^{1,3 1}Department 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. Nelson^1,2, David R. Hyde^{1 1}Department 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. Miller^{1,3 1}Department 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. Russell^{1 1}Department 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-Daescu^1,5, Gurpreet S. Sandhu^{2 1}Division 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-Porcel^{1 1}Divisions 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. Watzlawik^1,2,3, Robert J. Kahoud^1,3,5, Bharath Wootla^1,2,3, Meghan M. Painter^1,2,3, Arthur E. Warrington^1,2,3, William A. Carey^3,4, Moses Rodriguez^{1,2,3 1}Department 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-Daescu^2,5, Gurpreet S. Sandhu^{1 1}Division 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-Daescu^{1,4 1}Division 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. Macura^{2 1}Department 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. Su^{1 1}Department 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 Wijnen^4,5, Marianna Kruithof-de Julio^{1,6 1}Department 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 Zhu^{1,3 1}Department 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 Rodriguez^3,4, Sang-Hyun Oh^{1,2 1}Department 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 Jang^{1,3 1}Department 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 Farrugia^{1 1}Enteric 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. Knutson^{1 1}Department 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 Xu^{1 1}Division 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. Wengenack^{1 1}Department 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 1}Department 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. Schrum^{1 1}Department 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. Godwin^{1 1}Department 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.