University of Illinois, Chicago View Institution's Website 58 articles published in JoVE Medicine An Assay to Detect Protection of the Retinal Vasculature from Diabetes-Related Death in Mice Yanliang Li1, Andrius Kazlauskas1,2 1Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, 2Department of Physiology and Biophysics, University of Illinois at Chicago A protection assay was developed to monitor the retinal vasculature's resilience to death from diabetes/diabetic retinopathy-related insults such as oxidative stress and cytokines. Biology Comprehensive Echocardiographic Assessment of Right Ventricle Function in a Rat Model of Pulmonary Arterial Hypertension Paola C. Rosas*1, Liomar A. A. Neves*2, Peter B. Senese2, Michael R. Gralinski2 1Department of Pharmacy Practice, College of Pharmacy, University of Illinois at Chicago, 2CorDynamics, Inc. The present protocol describes the echocardiographic characterization of right ventricular morphology and function in a rat model of pulmonary arterial hypertension. Biology Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications Thanh Nguyen1, Sang Joon Ahn2, Richard West3, Ibra S. Fancher1 1Department of Kinesiology and Applied Physiology, College of Health Sciences, University of Delaware, 2Division of Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine, University of Illinois at Chicago, 3University of Delaware Flow Cytometry Core, Delaware Biotechnology Institute, University of Delaware This protocol details a method for the dissection of mouse adipose depots and the isolation and digestion of respective arteries to liberate and then identify the endothelial cell population. Freshly isolated cells used in downstream applications will advance the understanding of vascular cell biology and the mechanisms of vascular dysfunction. Medicine Echocardiographic Characterization of Left Ventricular Structure, Function, and Coronary Flow in Neonate Mice Shamim A. K. Chowdhury1, Paola C. Rosas1 1Department of Physiology and Biophysics, Center for Cardiovascular Research, College of Medicine, University of Illinois at Chicago The present protocol describes the echocardiographic assessment of left ventricular morphology, function, and coronary blood flow in 7-day old neonate mice. Biochemistry On-Chip Crystallization and Large-Scale Serial Diffraction at Room Temperature Linta M. Biju*1, Cong Wang*1, Weijia Kang1, Irin P. Tom1, Indika Kumarapperuma1, Xiaojing Yang1,2, Zhong Ren1,3 1Department of Chemistry, University of Illinois at Chicago, 2Department of Ophthalmology and Vision Sciences, University of Illinois at Chicago, 3Renz Research, Inc. This contribution describes how to set up protein crystallization on crystal-on-crystal devices and how to perform automated serial data collection at room temperature using the on-chip crystallization platform. Immunology and Infection Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals Tejabhiram Yadavalli1, Ipsita Volety1, Deepak Shukla1,2 1Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, 2Department of Microbiology and Immunology, University of Illinois at Chicago We describe the use of a porcine cornea to test the antiviral efficacy of experimental drugs. Medicine Taking the Next Step: a Neural Coaptation Orthotopic Hind Limb Transplant Model to Maximize Functional Recovery in Rat Feibo Zheng1,3, Andy Tully1,2, Kyle M Koss1, Xiaomin Zhang1, Longhui Qiu1, Jiao-Jing Wang1, Bilal A Naved1,4, David Z Ivancic1, James M Mathew1, Jason A Wertheim1, Zheng Jenny Zhang1 1Comprehensive Transplant Center and Department of Surgery, Feinberg School of Medicine, Northwestern University, 2Department of Surgery, University of Illinois at Chicago, 3Department of Surgery, Tianjin Occupational Diseases Precaution and Therapeutic Hospital, 4Department of Biomedical Engineering, McCormick School of Engineering, Northwestern University This protocol presents a robust, reproducible model of vascularized composite allotransplant (VCA) geared toward simultaneous study of immunology and functional recovery. The time invested in meticulous technique in a right mid-thigh hind limb orthotopic transplant with hand sewn vascular anastomoses and neural coaptation yields the ability to study functional recovery. Immunology and Infection A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections Dinesh Jaishankar1, Cormac Cosgrove1, Ryan J. Deaton2, I. Caroline Le Poole1,3 1Robert Lurie Comprehensive Cancer Center and Department of Dermatology, Feinberg School of Medicine, Northwestern University, 2Department of Pathology, University of Illinois at Chicago, 3Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University We describe a rapid staining method to perform multispectral imaging on frozen tissues. Biochemistry Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol Alexandria Slayden1, Kelsey North1, Shivantika Bisen1, Alex M. Dopico1, Anna N. Bukiya1, Avia Rosenhouse-Dantsker2 1Department of Pharmacology, Addiction Science and Toxicology, The University of Tennessee HSC, 2Department of Chemistry, University of Illinois at Chicago Two methods of cholesterol enrichment are presented: the application of cyclodextrin saturated with cholesterol to enrich mammalian tissues and cells, and the use of cholesterol-enriched phospholipid-based dispersions (liposomes) to enrich Xenopus oocytes. These methods are instrumental for determining the impact of elevated cholesterol levels in molecular, cellular, and organ function. Neuroscience Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia Yongchao Mou1,2, Sukhada Mukte1, Eric Chai1, Joshua Dein3, Xue-Jun Li1,2 1Department of Biomedical Sciences, University of Illinois College of Medicine Rockford, 2Department of Bioengineering, University of Illinois at Chicago, 3MD Program, University of Illinois College of Medicine Rockford Impaired mitochondrial transport and morphology are involved in various neurodegenerative diseases. The presented protocol uses induced pluripotent stem cell-derived forebrain neurons to assess mitochondrial transport and morphology in hereditary spastic paraplegia. This protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology, which will facilitate the study of neurodegenerative disease. Cancer Research Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures Wen-Yang Hu1, Dan-Ping Hu1, Lishi Xie1, Lynn A. Birch1, Gail S. Prins1,2,3 1Department of Urology, College of Medicine, University of Illinois at Chicago, 2Department of Pathology, College of Medicine, University of Illinois at Chicago, 3University of Illinois Cancer Center, College of Medicine, University of Illinois at Chicago Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS. Neuroscience Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum Shahin Shabanipour1,2, Azadeh Dalvand1,2, Xiaodan Jiao1,2, Maryam Rahimi Balaei1,2, Seung H. Chung3, Jiming Kong1, Marc. R. Del Bigio2,4, Hassan Marzban1,2 1Department of Human Anatomy and Cell Science, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, 2The Children's Hospital Research Institute of Manitoba (CHRIM), Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, 3Department of Oral Biology, University of Illinois at Chicago, 4Department of Pathology, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons. Biochemistry In Vivo Calcium Imaging in C. elegans Body Wall Muscles Ashley A. Martin1,2, Simon Alford3, Janet E. Richmond1 1Department of Biological Sciences, University of Illinois at Chicago, 2Department of Integrative Biology and Physiology, University of Minnesota, 3Department of Anatomy and Cell Biology, University of Illinois at Chicago This method provides a way to couple optogenetics and genetically encoded calcium sensors to image baseline cytosolic calcium levels and changes in evoked calcium transients in the body wall muscles of the model organism C. elegans. Immunology and Infection Electrophysiological Recordings of Single-cell Ion Currents Under Well-defined Shear Stress Ibra S. Fancher1, Irena Levitan1 1Department of Medicine, Division of Pulmonary, Critical Care, Sleep and Allergy, University of Illinois at Chicago The goal of this protocol is to describe a modified parallel plate flow chamber for use in investigating real time activation of mechanosensitive ion channels by shear stress. Biochemistry Using the Open-Source MALDI TOF-MS IDBac Pipeline for Analysis of Microbial Protein and Specialized Metabolite Data Chase M. Clark1, Maria S. Costa1,2, Erin Conley1, Emma Li1, Laura M. Sanchez1, Brian T. Murphy1 1Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 2Faculty of Pharmaceutical Sciences, University of Iceland IDBac is an open-source mass spectrometry-based bioinformatics pipeline that integrates data from both intact protein and specialized metabolite spectra, collected on cell material scraped from bacterial colonies. The pipeline allows researchers to rapidly organize hundreds to thousands of bacterial colonies into putative taxonomic groups, and further differentiate them based on specialized metabolite production. Cancer Research Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry Katherine E. Zink1, Matthew Dean1,2, Joanna E. Burdette1, Laura M. Sanchez1 1Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 2Department of Animal Science, University of Illinois at Urbana-Champaign A novel method of sample preparation was developed to accommodate cell and tissue coculture to detect small molecule exchange using imaging mass spectrometry. Bioengineering Handling and Assessment of Human Primary Prostate Organoid Culture Tara McCray*1, Zachary Richards*1, Joseph Marsili1, Gail S. Prins1,2,3, Larisa Nonn1,3 1Department of Pathology, University of Illinois at Chicago, 2Departments of Urology, Physiology, and Biophysics, University of Illinois at Chicago, 3University of Illinois Cancer Center Here, we present a protocol to guide human primary prostate organoid handling then suggest endpoints to assess phenotype. Seeding, culture maintenance, recovery from matrix gel, morphologic quantification, embedding and sectioning, FFPE sectioning, whole-mount staining, and application of commercial assays are described. Cancer Research Development and Angiographic Use of the Rabbit VX2 Model for Liver Cancer Ramzy C. Khabbaz1,2, Yu-Hui Huang1,2, Alexandra A. Smith3, Kelly D. Garcia1, R. Peter Lokken1, Ron C. Gaba1 1Department of Radiology, University of Illinois, 2College of Medicine, University of Illinois, 3Department of Biological Resources Laboratory, University of Illinois The goal of this article is to provide a primer for the development and use of the VX2 carcinoma rabbit model for liver cancer. Neuroscience Quantifying Acute Changes in Renal Sympathetic Nerve Activity in Response to Central Nervous System Manipulations in Anesthetized Rats Anne M. Fink1, Caron Dean2 1Department of Biobehavioral Health Science, College of Nursing, University of Illinois at Chicago, 2Department of Anesthesiology, Medical College of Wisconsin and Zablocki VA Medical Center Methods for measuring sympathetic and cardiovascular responses to central nervous system (CNS) manipulations are important for advancing neuroscience. This protocol was developed to assist scientists with measuring and quantifying acute changes in renal sympathetic nerve activity (RSNA) in anesthetized rats (non-survival). Neuroscience Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes Rafael E. Flores-Obando1, Mona M. Freidin2, Charles K. Abrams2 1Program in Molecular and Cellular Biology, State University of New York Downstate Medical Center, 2Department of Neurology and Rehabilitation, University of Illinois at Chicago We describe the immunomagnetic isolation of primary mouse oligodendrocytes, which allows the rapid and specific isolation of the cells for in vitro culture. Bioengineering Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro Corey M. Rountree1, John B. Troy2, Laxman Saggere1 1Department of Mechanical and Industrial Engineering, University of Illinois at Chicago, 2Department of Biomedical Engineering, Northwestern University This protocol describes a novel method for investigating a form of chemical neurostimulation of wholemount rat retinas in vitro with the neurotransmitter glutamate. Chemical neurostimulation is a promising alternative to the conventional electrical neurostimulation of retinal neurons for treating irreversible blindness caused by photoreceptor degenerative diseases. Medicine Esophageal Heat Transfer for Patient Temperature Control and Targeted Temperature Management Melissa I. Naiman1,2, Maria Gray2, Joseph Haymore3, Ahmed F. Hegazy4, Andrej Markota5, Neeraj Badjatia6, Erik B. Kulstad2,7 1Center for Advanced Design, Research, and Exploration, University of Illinois at Chicago, 2Attune Medical, 3University of Maryland School of Nursing, 4University of Western Ontario, 5University Medical Centre Maribor, 6University of Maryland, 7Department of Emergency Medicine, University of Texas, Southwestern Medical Center This study presents a novel method to provide efficient patient temperature control for cooling or warming patients. A single use, triple lumen device is placed into the esophagus, analogous to a standard orogastric tube, and connects to existing heat exchange units to perform automatic patient temperature management. Medicine In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption Riya Thomas1, Kazandra Diaz1, Kevin P. Koster1, Leon M. Tai1 1Department of Anatomy and Cell Biology, University of Illinois at Chicago Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage. Engineering High-resolution Thermal Micro-imaging Using Europium Chelate Luminescent Coatings Timothy M. Benseman1,2,3, Yang Hao1,2, Vitalii K. Vlasko-Vlasov1, Ulrich Welp1, Alexei E. Koshelev1, Wai-Kwong Kwok1, Ralu Divan4, Courtney Keiser5, Chiharu Watanabe6, Kazuo Kadowaki6 1Materials Science Division, Argonne National Laboratory, 2Department of Physics, University of Illinois at Chicago, 3Department of Physics, CUNY Queens College, 4Center for Nanoscale Materials, Argonne National Laboratory, 5Department of Physics, University of Northern Iowa, 6Institute for Materials Science, University of Tsukuba Europium thenoyltrifluoroacetonate (EuTFC) has an optical luminescence line at 612 nm, whose activation efficiency decreases strongly with temperature. If a sample coated with a thin film of this material is micro-imaged, the 612 nm luminescent response intensity may be converted into a direct map of sample surface temperature. Biology Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D Arivarasu N. Anabazhagan*1, Ishita Chatterjee*1, Shubha Priyamvada1, Anoop Kumar1, Sangeeta Tyagi1, Seema Saksena1,2, Waddah A. Alrefai1,2, Pradeep K. Dudeja1,2, Ravinder K. Gill1 1Department of Medicine, University of Illinois at Chicago, 2Department of Research, Jesse Brown VA Medical Center We describe simple methods to study the regulation of intestinal serotonin transporter (SERT) function and expression using an in vitro cell culture model of Caco-2 cells grown in 3D and an ex vivo model of mouse intestines. These methods are applicable to the study of other epithelial transporters. Cancer Research Synthesis and Characterization of an Aspirin-fumarate Prodrug that Inhibits NFκB Activity and Breast Cancer Stem Cells Irida Kastrati1, Loruhama Delgado-Rivera2, Gergana Georgieva3, Gregory R. J. Thatcher2, Jonna Frasor1 1Physiology and Biophysics, College of Medicine, University of Illinois at Chicago, 2Medicinal Chemistry and Pharmacognosy, College of Medicine, University of Illinois at Chicago, 3College of Pharmacy, University of Illinois at Chicago This procedure will demonstrate how we synthesized and characterized the anti-NFκB and anti-cancer stem cell activity of an aspirin-fumarate prodrug. Biology Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death Snezana Vujicic*1,2, Lanfei Feng*1,2, Angelika Antoni3, Joyce Rauch4, Jerrold S. Levine1,2,5 1Section of Nephrology, Department of Medicine, University of Illinois at Chicago, 2Section of Nephrology, Department of Medicine, Jesse Brown Veterans Affairs Medical Center, 3Department of Biology, Kutztown University of Pennsylvania, 4Division of Rheumatology, Department of Medicine, Research Institute of the McGill University Health Centre, 5Department of Microbiology & Immunology, University of Illinois at Chicago Here, we present a protocol for the determination of intracellular signaling events induced in viable cells by physical interaction with adjacent dead or dying cells. The protocol focuses on signaling events induced by receptor-mediated recognition of the dead cells, as opposed to their phagocytic uptake or release of soluble mediators. Biochemistry An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides Yi-Wen Hsieh1, Amel Alqadah1, Chiou-Fen Chuang1 1Department of Biological Sciences, University of Illinois at Chicago We describe here an optimized protocol of fluorescent Electrophoretic Mobility Shift Assays (fEMSA) using purified SOX-2 proteins together with infrared fluorescent dye-labeled DNA probes as a case study to tackle an important biological question. Medicine A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse Neda Afsharkhamseh1, Elham Ghahari1, Medi Eslani1, Ali R. Djalilian1 1Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago The following article provides an easy, reproducible technique to effectively create a sustainable mouse model of limbal stem cell deficiency (LSCD). This animal model is useful in testing and comparing the efficacy of treatments for limbal stem cell diseases. Medicine A Novel Approach for the Administration of Medications and Fluids in Emergency Scenarios and Settings Akilesh Honasoge1, Neal Lyons2, Kathleen Hesse3, Braden Parker3, Robert Mokszycki2, Kelly Wesselhoff3, Rolla Sweis2, Erik B. Kulstad3 1College of Medicine, University of Illinois at Chicago, 2Department of Pharmacy, Advocate Christ Medical Center, 3Department of Emergency Medicine, Advocate Christ Medical Center This study presents a novel device that offers an easy route to quickly provide medications and fluids in patients with limited or difficult IV access. This small-diameter device is placed in the distal third of the rectum, allowing for ongoing medication and fluids administration. Medicine Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis Giovanni Lugli1, Yachana Kataria1, Zachary Richards1, Peter Gann1, Xiaofeng Zhou2, Larisa Nonn1 1Department of Pathology, University of Illinois at Chicago, 2Department of Periodontics, University of Illinois at Chicago The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis. Immunology and Infection Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes P. David McMullen1, Nancy E. Freitag1 1Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals. Medicine Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury Deborah N. Olmstead1,2, Nichole A. Mesnard-Hoaglin3, Richard J. Batka1,2, Melissa M. Haulcomb1,2, Whitney M. Miller1,2, Kathryn J. Jones1,2 1Anatomy and Cell Biology, Indiana University School of Medicine, 2Research and Development Services, Richard L. Roudebush VA Medical Center, 3Department of Anatomy and Cell Biology, University of Illinois, Chicago We present a surgical protocol detailing how to perform a cut or crush axotomy on the facial nerve in the mouse. The facial nerve axotomy can be employed to study the physiological response to nerve injury and test therapeutic techniques. Medicine High-definition Fourier Transform Infrared (FT-IR) Spectroscopic Imaging of Human Tissue Sections towards Improving Pathology Hari Sreedhar*1, Vishal K. Varma*1, Peter L. Nguyen2, Bennett Davidson3,4, Sanjeev Akkina5, Grace Guzman2, Suman Setty2, Andre Kajdacsy-Balla2, Michael J. Walsh2 1Department of Bioengineering, University of Illinois at Chicago, 2Department of Pathology, University of Illinois at Chicago, 3Department of Biological Sciences, University of Illinois at Chicago, 4Department of Chemistry, University of Illinois at Chicago, 5Department of Nephrology, University of Illinois at Chicago Fourier Transform Infrared (FT-IR) spectroscopic imaging is a fast and label-free approach to obtain biochemical data sets of cells and tissues. Here, we demonstrate how to obtain high-definition FT-IR images of tissue sections towards improving disease diagnosis. Neuroscience A Method of Nodose Ganglia Injection in Sprague-Dawley Rat Michael W. Calik1,3, Miodrag Radulovacki1,2, David W. Carley1,3 1Center for Narcolepsy, Sleep and Health Research, University of Illinois at Chicago, 2Department of Pharmacology, University of Illinois at Chicago, 3Department of Biobehavioral Health Science, University of Illinois at Chicago Afferent vagal signaling transmits important information to central nervous system from receptors located in organs of the abdomen and thorax. The nodose ganglia of vagus nerves contain many types of receptors that modulate vagal activity. This protocol describes a method of local injections of neurochemicals into the nodose ganglia. Immunology and Infection Pseudomonas aeruginosa Induced Lung Injury Model Varsha Suresh Kumar1, Ruxana T. Sadikot2, Jeanette E. Purcell3, Asrar B. Malik1, Yuru Liu1 1Department of Pharmacology, University of Illinois College of Medicine, University of Illinois at Chicago, 2Section of Pulmonary and Critical Care Medicine, Atlanta VAMC, Emory University, 3Biologic Resources Lab, University of Illinois at Chicago We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant. Biology The Soft Agar Colony Formation Assay Stanley Borowicz1, Michelle Van Scoyk2, Sreedevi Avasarala2, Manoj Kumar Karuppusamy Rathinam2, Jordi Tauler2, Rama Kamesh Bikkavilli2, Robert A. Winn2,3 1Department of Hematology and Oncology, University of Illinois at Chicago, 2Department of Pulmonary, Critical Care, Sleep, and Allergy, University of Illinois at Chicago, 3Jesse Brown Veterans Affairs Medical Center The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells. Neuroscience Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell Kelly T. Varga1, Zhongjiao Jiang1, Liang-Wei Gong1 1Department of Biological Sciences, University of Illinois at Chicago After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells. Neuroscience An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy Kumiko I. Claycomb1, Kasey M. Johnson1, Ernesto R. Bongarzone2, Stephen J. Crocker1 1Department of Neuroscience, University of Connecticut Health Center, 2Department of Anatomy and Cell Biology, University of Illinois at Chicago Globoid cells are a defining pathological feature of Krabbe disease, a leukodystrophy currently lacking an effective long-term therapy. We have developed a cell culture model to study the innate biology and pathogenic potential of activated microglia and their transformation into globoid cells. Biology In vitro Methylation Assay to Study Protein Arginine Methylation Rama Kamesh Bikkavilli1, Sreedevi Avasarala1, Michelle Van Scoyk1, Manoj Kumar Karuppusamy Rathinam1, Jordi Tauler1, Stanley Borowicz1,2, Robert A. Winn1,3 1Department of Pulmonary, Critical Care, Sleep, and Allergy, University of Illinois at Chicago, 2Department of Hematology and Oncology, University of Illinois at Chicago, 3Jesse Brown Veterans Affairs Medical Center Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates. Neuroscience Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals Shankar Ramachandran1, Simon Alford1 1Biological Sciences, University of Illinois at Chicago Recording Ca2+ currents at the presynaptic release face membrane is key to a precise understanding of Ca2+ entry and neurotransmitter release. We present an acute dissociation of the lamprey spinal cord that yields functional isolated reticulospinal axons, permitting recording directly from the release face membrane of individual presynaptic terminals. Behavior Using Eye Movements to Evaluate the Cognitive Processes Involved in Text Comprehension Gary E. Raney1, Spencer J. Campbell1, Joanna C. Bovee1 1Department of Psychology, University of Illinois at Chicago The present article describes how to use eye tracking methodologies to study the cognitive processes involved in text comprehension. Descriptions of eye tracking equipment, how to develop experimental stimuli, and procedural recommendations are included. The information presented can be applied to most any study using verbal stimuli. Bioengineering Quantitative and Temporal Control of Oxygen Microenvironment at the Single Islet Level Joe Fu-Jiou Lo1, Yong Wang2,3, Zidong Li1, Zhengtuo Zhao1, Di Hu1, David T. Eddington3, Jose Oberholzer2,3 1Department of Mechanical Engineering, University of Michigan-Dearborn, 2Department of Surgery/Transplant, University of Illinois at Chicago, 3Department of Bioengineering, University of Illinois at Chicago Microfluidic oxygen control confers more than just convenience and speed over hypoxic chambers for biological experiments. Especially when implemented via diffusion through a membrane, microfluidic oxygen can provide simultaneous liquid and gas phase modulations at the microscale-level. This technique enables dynamic multi-parametric experiments critical for studying islet pathophysiology. Engineering Characterization of Electrode Materials for Lithium Ion and Sodium Ion Batteries Using Synchrotron Radiation Techniques Marca M. Doeff1, Guoying Chen1, Jordi Cabana1,2, Thomas J. Richardson1, Apurva Mehta3, Mona Shirpour1, Hugues Duncan1, Chunjoong Kim1, Kinson C. Kam4, Thomas Conry5 1Environmental Energy Technologies Division, Lawrence Berkeley National Laboratory, 2Department of Chemistry, University of Illinois at Chicago, 3Stanford Synchrotron Radiation Lightsource, 4Haldor Topsøe A/S, 5PolyPlus Battery Company We describe the use of synchrotron X-ray absorption spectroscopy (XAS) and X-ray diffraction (XRD) techniques to probe details of intercalation/deintercalation processes in electrode materials for Li-ion and Na-ion batteries. Both in situ and ex situ experiments are used to understand structural behavior relevant to the operation of devices Biology Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction Li Wang1, Graeme K. Carnegie1 1Department of Pharmacology, University of Illinois at Chicago Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction. Immunology and Infection Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice Kyung Ho Kim1, Andrew Barazia1, Jaehyung Cho1,2 1Department of Pharmacology, University of Illinois at Chicago, 2Department of Anesthesiology, University of Illinois at Chicago Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions. Bioengineering Haptic/Graphic Rehabilitation: Integrating a Robot into a Virtual Environment Library and Applying it to Stroke Therapy Ian Sharp1, James Patton1, Molly Listenberger2, Emily Case2 1Department of Bioengineering, University of Illinois at Chicago and Rehabilitation Institute of Chicago, 2Sensory Motor Performance Program, Rehabilitation Institute of Chicago Recently, a vast amount of prospects have come available for human-robot interactive systems. In this paper we outline the integration of a new robotic device with open source software that can rapidly make possible a library of interactive functionality. We then outline a clinical application for a neurorehabilitation application. Bioengineering Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts Shelby M. King*1, Suzanne Quartuccio*1, Tyvette S. Hilliard*1, Kari Inoue1, Joanna E. Burdette1 1Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation. Medicine Methods to Quantify Pharmacologically Induced Alterations in Motor Function in Human Incomplete SCI Christopher K. Thompson1,2, Arun Jayaraman1, Catherine Kinnaird1, T. George Hornby1,3 1Sensory Motor Performance Program, Rehabilitation Institute of Chicago, 2Department of Kinesiology and Nutrition, University of Illinois at Chicago, 3Department of Physical Therapy, University of Illinois at Chicago This video demonstrates modulation of reflex activity, volitional strength and ambulation through clinical and quantitative assessments in individuals with motor incomplete SCI as a result of acute oral administration of a serotonin reuptake inhibitor (SSRI). Biology Bimolecular Fluorescence Complementation Katy A. Wong1, John P. O'Bryan1 1Department of Pharmacology, University of Illinois at Chicago The subcellular localization of proteins is important in determining the spatio-temporal regulation of cell signaling. Here, we describe bimolecular fluorescence complementation (BiFC) as a straightforward method for monitoring the spatial interactions of proteins in the cell. Biology Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein Forum H. Bhatt1, Constance J. Jeffery1 1Department of Biological Sciences, University of Illinois Chicago - UIC In this protocol we demonstrate the expression, solubilization, and purification of a recombinantly expressed membrane protein, MexB, as a soluble protein detergent complex. MexB is a multidrug resistance membrane transporter from the opportunistic bacterial pathogen Pseudomonas aeruginosa. Biology Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets Michael L. Beshiri1, Abul Islam2, Dannielle C. DeWaal1, William F. Richter1, Jennifer Love3, Nuria Lopez-Bigas2, Elizaveta V. Benevolenskaya1 1Department of Biochemistry and Molecular Genetics, University of Illinois Chicago - UIC, 2Research Unit on Biomedical Informatics, Universitat Pompeu Fabra, 3Genome Technology Core, Whitehead Institute for Biomedical Research Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase. Biology A Multi-Parametric Islet Perifusion System within a Microfluidic Perifusion Device Adeola F. Adewola1, Yong Wang1, Tricia Harvat1, David T. Eddington2, Dongyoung Lee1, Jose Oberholzer1,2 1Department of Surgery, University of Illinois, Chicago, 2Department of Bioengineering, University of Illinois, Chicago A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence imaging of calcium influx and mitochondrial potential changes. Biology Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue Meirigeng Qi1, Barbara Barbaro1, Shusen Wang1, Yong Wang1, Mike Hansen1, Jose Oberholzer1 1Department of Surgery, University of Illinois, Chicago Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method. Biology Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets Meirigeng Qi1, Barbara Barbaro1, Shusen Wang1, Yong Wang1, Mike Hansen1, Jose Oberholzer1 1Department of Surgery, University of Illinois, Chicago Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method. Biology Dissecting and Recording from The C. Elegans Neuromuscular Junction Janet Richmond1 1Department of Biological Sciences, University of Illinois, Chicago Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied. Biology Applying Microfluidics to Electrophysiology David T. Eddington1 1Dept. of Bioengineering, University of Illinois, Chicago Biology Brain Slice Stimulation Using a Microfluidic Network and Standard Perfusion Chamber Javeed Shaikh Mohammed1, Hugo Caicedo1, Christopher P. Fall2, David T. Eddington1 1Dept. of Bioengineering, University of Illinois, Chicago, 2Department of Anatomy and Cell Biology, University of Illinois, Chicago We demonstrate fabrication of a simple microfluidic device that can be integrated with standard electrophysiology setups to expose microscale surfaces of a brain slice in a well controlled manner to different neurotransmitters.
