University of Alabama, Birmingham View Institution's Website 60 articles published in JoVE Medicine Radiation Planning Assistant - A Web-based Tool to Support High-quality Radiotherapy in Clinics with Limited Resources Laurence Edward Court1, Ajay Aggarwal2, Hester Burger3, Carlos Cardenas4, Christine Chung1, Raphael Douglas1, Monique du Toit5, Anuja Jhingran1, Raymond Mumme1, Sikudhani Muya6, Komeela Naidoo5, Jerry Ndumbalo6, Tucker Netherton1, Callistus Nguyen1, Adenike Olanrewaju1, Jeannette Parkes3, Willie Shaw7, Christoph Trauernicht5, Melody Xu8, Jinzhong Yang1, Lifei Zhang1, Hannah Simonds9, Beth M. Beadle10 1The University of Texas MD Anderson Cancer Center, 2Guy’s and St. Thomas’ Hospital, 3Groote Schuur Hospital and University of Cape Town, 4University of Alabama at Birmingham, 5Tygerberg Hospital and Stellenbosch University, 6Ocean Road Cancer Institute, 7University of the Free State, 8University of California-San Francisco, 9University Hospitals Plymouth NHS Trust, 10Stanford University This protocol describes a series of automated tools designed for high-quality radiotherapy autocontouring and autoplanning that are being packaged into a web-based service to maximize robustness and scalability while minimizing operational costs. Medicine A Workflow to Quantitatively Determine Age-Related Macular Degeneration Lesion-Specific Variations in Fundus Autofluorescence Leon von der Emde*1, Merten Mallwitz*1, Frank G. Holz1, Kenneth R. Sloan2, Thomas Ach1 1Department of Ophthalmology, University of Bonn, 2Department of Ophthalmology and Visual Sciences, University of Alabama at Birmingham This research describes a workflow to determine and compare autofluorescence levels from individual regions of interest (e.g., drusen and subretinal drusenoid deposits in age-related macular degeneration [AMD]) while accounting for varying autofluorescence levels throughout the fundus. Biology Isolation of Retinal Pigment Epithelial Cells from Guinea Pig Eyes So Goto1,2,3, Michael Frost4, Christine Wildsoet1 1Herbert Wertheim School Optometry and Vision Science, University of California, Berkeley, 2Department of Ophthalmology, Osaka University Graduate School of Medicine, 3Department of Ophthalmology, Tokyo Medical Center, National Hospital Organization, 4Department of Optometry and Vision Science, School of Optometry, University of Alabama at Birmingham We describe a simple and efficient method for isolating cells of the retinal pigment epithelium (RPE) cells from the eyes of young pigmented guinea pigs. This procedure allows for follow-up molecular biology studies on the isolated RPE, including gene expression analyses. Editorial Novel Translational Approaches To Study Kidney Disease Emma Laurence1, Parveen Kumar1, Tanecia Mitchell1 1Department of Urology, University of Alabama at Birmingham Biology Tension Gauge Tether Probes for Quantifying Growth Factor Mediated Integrin Mechanics and Adhesion Tejeshwar C. Rao1, Alexa L. Mattheyses1 1Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham TGT surface is an innovative platform to study growth factor-integrin crosstalk. The flexible probe design, specificity of the adhesion ligand, and precise modulation of stimulation conditions allow robust quantitative assessments of EGFR-integrin interplay. The results highlight EGFR as a 'mechano-organizer' tuning integrin mechanics, influencing focal adhesion assembly and cell spreading. Medicine Culture and Imaging of Human Nasal Epithelial Organoids Zhongyu Liu1,2, Justin D. Anderson1,2, Jennifer Natt1,2, Jennifer S. Guimbellot1,2 1Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham (UAB), 2Department of Pediatrics, Division of Pulmonary and Sleep Medicine, UAB A detailed protocol is presented here to describe an in vitro organoid model from human nasal epithelial cells. The protocol has options for measurements requiring standard laboratory equipment, with additional possibilities for specialized equipment and software. Cancer Research Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells Jody Fromm Longo1, Stephanie N. Brosius2,3, Steven L. Carroll1 1Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 2Department of Pediatrics and Neurology, Perelman School of Medicine of the University of Pennsylvania, 3Division of Child Neurology, Children's Hospital of Philadelphia Here, we present a protocol for performing gene knockouts that are embryonic lethal in vivo in genetically engineered mouse model-derived tumors and then assessing the effect that the knockout has on tumor growth, proliferation, survival, migration, invasion, and the transcriptome in vitro and in vivo. Medicine Segmentation and Linear Measurement for Body Composition Analysis using Slice-O-Matic and Horos Sean Steele1, Fangyi Lin1, Thien-Linh Le1, Alexandra Medline1, Michelle Higgins1, Alex Sandberg1, Sean Evans1, Gordon Hong2, Milton A. Williams3, Mehmet A. Bilen4, Sarah Psutka5, Kenneth Ogan1, Viraj A. Master1 1Department of Urology, Emory University School of Medicine, 2Northeast Ohio Medical University, 3Department of Urology, University of Alabama at Birmingham, 4Department of Medical Oncology, Winship Cancer Institute of Emory University, 5Department of Urology, University of Washington Segmentation and linear measurements quantify skeletal muscle mass and adipose tissues using Computed Tomography and/or Magnetic Resonance Imaging images. Here, we outline the use of Slice-O-Matic software and Horos image viewer for rapid and accurate analysis of body composition. These methods can provide important information for prognosis and risk stratification. Medicine Estimation of Urinary Nanocrystals in Humans using Calcium Fluorophore Labeling and Nanoparticle Tracking Analysis Parveen Kumar1, Andrew Bell1, Tanecia Mitchell1 1Department of Urology, University of Alabama at Birmingham The objective of this study was to determine whether nanoparticle tracking analysis (NTA) could detect and quantify urinary calcium containing nanocrystals from healthy adults. The findings from the current study suggest NTA could be a potential tool to estimate urinary nanocrystals during kidney stone disease. Cancer Research Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone Blake E. Hildreth III1, Charlotte Palmer2, Matthew J. Allen2 1Department of Pathology and O'Neal Comprehensive Cancer Center, University of Alabama at Birmingham, 2Surgical Discovery Centre, Department of Veterinary Medicine, University of Cambridge Bone metastasis models do not develop metastasis uniformly or with a 100% incidence. Direct intra-osseous tumor cell injection can result in embolization of the lung. We present our technique modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone, leading to reproducible engraftment and growth. Behavior Fear Incubation Using an Extended Fear-Conditioning Protocol for Rats César Acevedo-Triana1,6, Javier L. Rico2, Leonardo A. Ortega2, Melissa Andrea N. Cardenas3, Fernando P. Cardenas3, Manuel J. Rojas4, Juan Carlos Forigua-Vargas2, Julián Cifuentes2, Camilo Hurtado-Parrado2,5 1School of Psychology, Universidad Pedagógica y Tecnológica de Colombia, 2Animal Behavior Laboratory, Fundación Universitaria Konrad Lorenz, 3Department of Psychology, Universidad de Los Andes, 4School of Veterinary Medicine, Animal Health Department, Universidad Nacional de Colombia, 5Department of Psychology, Troy University, 6Department of Neurobiology, University of Alabama at Birmingham We describe an extended fear-conditioning protocol that produces overtraining and fear incubation in rats. This protocol entails a single training session with 25 tone-shock pairings (i.e., overtraining) and a comparison of conditioned freezing responses during context and cue tests 48 h (short-term) and 6 weeks (long-term) after training. Cancer Research Perfusion and Inflation of the Mouse Lung for Tumor Histology Mackenzie L. Davenport1, Taylor P. Sherrill2, Timothy S. Blackwell2, Mick D. Edmonds1 1Department of Genetics, University of Alabama at Birmingham, 2Department of Medicine, Division of Allergy, Pulmonary, and Critical Care Medicine, Vanderbilt University School of Medicine The purpose of this method is to present a simple and efficient method for the perfusion, inflation, and fixation of mouse lungs for the examination of lung tumor pathology and evaluation of metastases to the lung. Neuroscience A Benchtop Approach to the Location Specific Blood Brain Barrier Opening using Focused Ultrasound in a Rat Model Megan Rich1, Quentin Whitsitt1, Farah Lubin2, Mark Bolding1 1Department of Radiology, University of Alabama at Birmingham, 2Department of Neurobiology, University of Alabama at Birmingham Focused ultrasound with microbubble agents can open the blood brain barrier focally and transiently. This technique has been used to deliver a wide range of agents across the blood brain barrier. This article provides a detailed protocol for the localized delivery to the rodent brain with or without MRI guidance. Medicine Measurement of Natural Killer Cell-Mediated Cytotoxicity and Migration in the Context of Hepatic Tumor Cells Suresh Chava1, Suresh Bugide1, Romi Gupta1, Narendra Wajapeyee1 1Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham Evasion of natural killer (NK) cell-mediated eradication by cancer cells is important for cancer initiation and progression. Here, we present two non-radioactivity-based protocols to evaluate NK cell-mediated cytotoxicity toward hepatic tumor cells. Additionally, a third protocol is presented to analyze NK cell migration. Medicine Inguinal Subcutaneous White Adipose Tissue (ISWAT) Transplantation Model of Murine Islets Yuanzheng Peng*1, Zhicheng Zou*1, Jiao Chen1, Hancheng Zhang1, Ying Lu1, Rito Bittino2, Hongxing Fu3, David K. C. Cooper4, Shan Lin1, Mengtao Cao1, Yifan Dai5, Zhiming Cai1, Lisha Mou1 1Shenzhen Xenotransplantation Medical Engineering Research and Development Center, Institute of Translational Medicine, Shenzhen Second People's Hospital, First Affiliated Hospital of Shenzhen University, Health Science Center, 2Institute for Cellular Therapeutics, Allegheny-Singer Research Institute, 3School of Pharmaceutical Sciences, Wenzhou Medical University, 4Xenotransplantation Program/Department of Surgery, University of Alabama at Birmingham, 5Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University In this protocol, a method of murine islet isolation and transplantation into the inguinal subcutaneous white adipose tissue is described. Isolated syngeneic murine islets are transplanted into a murine recipient using a basement membrane hydrogel. The blood glucose level of the recipients is monitored, and histology analysis of the islet grafts is performed. Developmental Biology A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals Courtney Barkley1, Rosa Serra1, Sarah B. Peters1 1Cell, Developmental and Integrative Biology Department, University of Alabama at Birmingham We describe the isolation, dispersion and plating of dental pulp (DP) primary cells with trigeminal (TG) neurons cultured atop overlying transwell filters. Cellular responses of DP cells can be analyzed with immunofluorescence or RNA/protein analysis. Immunofluorescence of neuronal markers with confocal microscopy permits the analysis of neurite outgrowth responses. Cancer Research Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports Kelly Pittman1, Shelton Earp1, Eric Ubil2 1Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, 2O'Neal Comprehensive Cancer Center, University of Alabama at Birmingham Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation. Medicine Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts Luther M. Swift1,2, Rafael Jaimes III1,2, Damon McCullough1,2, Morgan Burke1,2, Marissa Reilly1,2, Takuya Maeda1,2,3, Hanyu Zhang4, Nobuyuki Ishibashi1,2,3, Jack M. Rogers4, Nikki Gillum Posnack1,2,5 1Center for Neuroscience Research, Children's National Hospital, 4Department of Biomedical Engineering, School of Engineering, University of Alabama at Birmingham, 5Department of Pediatrics, Department of Pharmacology & Physiology, School of Medicine and Health Sciences, George Washington University The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model. Biology Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs Yanli Zhao1,2,3, Chengjiang Zhao4, David K.C. Cooper5, Ying Lu2, Kewang Luo6, Huiyun Wang3, Pengfei Chen3, Changchun Zeng3, Shaodong Luan1, Lisha Mou2, Hanchao Gao1,2,3 1Department of Nephrology, Shenzhen Longhua District Central Hospital, Affiliated Central Hospital of Shenzhen Longhua District, Guangdong Medical University, 2Shenzhen Xenotransplantation Medical Engineering Research and Development Center, Shenzhen Second People's Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen University School of Medicine, 3Department of Medical Laboratory, Shenzhen Longhua District Central Hospital, Affiliated Central Hospital of Shenzhen Longhua District, Guangdong Medical University, 4Department of Endocrinology, Shenzhen Longhua District Central Hospital, Affiliated Central Hospital of Shenzhen Longhua District, Guangdong Medical University, 5Xenotransplantation Program, Department of Surgery, University of Alabama at Birmingham, 6 An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation. Biochemistry Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity Meng Zhao1, Yawen Tang1, Patrick J. Ernst1,2, Asher Kahn-Krell1, Chengming Fan1,3, Danielle Pretorius1, Hanxi Zhu1, Xi Lou1, Lufang Zhou2, Jianyi Zhang1, Wuqiang Zhu1 1Department of Biomedical Engineering, School of Medicine, School of Engineering, University of Alabama at Birmingham, 2Division of Cardiovascular Diseases, School of Medicine, School of Engineering, University of Alabama at Birmingham, 3Department of Cardiac Surgery, The Second Xiangya Hospital of Central South University In this protocol, we demonstrate and elaborate on how to use human induced pluripotent stem cells for cardiomyocyte differentiation and purification, and further, on how to improve its transplantation efficiency with Rho-associated protein kinase inhibitor pretreatment in a mouse myocardial infarction model. Developmental Biology Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract Muhan Hu1, Shara Legg1, Michael A. Miller1 1Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham Sperm must successfully navigate through the oviduct to fertilize an oocyte. Here, we describe an assay for measuring sperm migration within the C. elegans hermaphrodite uterus. This assay can provide quantitative data on sperm distribution within the uterus after mating, as well as on speed, directional velocity, and reversal frequency. Neuroscience Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo Joseph R. Patterson1, Nicole K. Polinski2, Megan F. Duffy1,3, Christopher J. Kemp1, Kelvin C. Luk4, Laura A. Volpicelli-Daley5, Nicholas M. Kanaan1, Caryl E. Sortwell1,3,6 1Department of Translational Science and Molecular Medicine, Michigan State University, 2 The goal of this article is to outline the steps required for the generation of fibrils from monomeric alpha-synuclein, subsequent quality control, and use of the preformed fibrils in vivo. Biochemistry Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo Patrick Moran1,2, Yichen Guo3,4, Rong Yuan1,3, Nicholas Barnekow1, Jordan Palmer2, Adam Beck3, Bin Ren3,4,5 1Blood Research Institute, Blood Center of Wisconsin, 2Department of Medicine, Medical College of Wisconsin, 3Department of Surgery, The University of Alabama at Birmingham, 4Department of Biomedical Engineering, The University of Alabama at Birmingham, 5GBS Program, Graduate School, The University of Alabama at Birmingham We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification. Immunology and Infection A High-throughput Shigella-specific Bactericidal Assay Hailey P. Weerts1, Jigui Yu2, Robert W. Kaminski1, Moon H. Nahm2 1Department of Enteric Infections, Bacterial Diseases Branch, Walter Reed Army Institute of Research, 2University of Alabama at Birmingham Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer. Genetics Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy Jessica M. Miller*1, Nikhil Maneesh Mardhekar*1, Vasanthi Rajasekaran1, Jianyi Zhang1, Ram Kannappan1 1Department of Biomedical Engineering, School of Engineering, School of Medicine, University of Alabama at Birmingham Here, we provide a detailed description of an experimental setup for an analysis of the assessment of DNA integrity in stem cells prior to cell transplantation. Immunology and Infection Assaying for Inorganic Polyphosphate in Bacteria Arya Pokhrel1, Jordan C. Lingo2, Frank Wolschendorf2, Michael J. Gray1 1Department of Microbiology, University of Alabama at Birmingham, 2Division of Infectious Diseases, University of Alabama at Birmingham We describe a simple method for rapid quantification of inorganic polyphosphate in diverse bacteria, including Gram-negative, Gram-positive, and mycobacterial species. Biology A Method for Islet Transplantation to the Omentum in Mouse Ying Lu*1, Shangyou Zou*1, Suzanne Bertera*2, Rita Bottino2, David K.C. Cooper3, Zhengzhao Liu1, Yi Huang1, Chengjun Wang1, Chungu Hong1, Tian He1, Hancheng Zhang1, Qin Huo4, Hongxing Fu5, Zhiming Cai1, Lisha Mou1 1Shenzhen Xenotransplantation Medical Engineering Research and Development Center, Institute of Translational Medicine, Shenzhen Second People's Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen University School of Medicine, Shenzhen University Health Science Center, 2Institute for Cellular Therapeutics, Allegheny-Singer Research Institute, 3Xenotransplantation Program/Department of Surgery, The University of Alabama at Birmingham, 4College of Life Science and Oceanography, Shenzhen University, 5School of Pharmcy, Wenzhou Medical College A method for the omental transplantation of islets in a mouse is introduced. The isolated islets are mixed with hydrogel and the mixture is placed into the omental pouch of a diabetic mouse. Then, the blood glucose is monitored, and immuno-histochemical analysis is performed. Medicine Standardized Measurement of Nasal Membrane Transepithelial Potential Difference (NPD) George M. Solomon1, Inez Bronsveld2, Kathryn Hayes3, Michael Wilschanski4, Paola Melotti5, Steven M. Rowe1, Isabelle Sermet-Gaudelus6,7 1Department of Medicine and the Gregory Fleming James Cystic Fibrosis Center, University of Alabama at Birmingham, 2Department of Pulmonology and Tuberculosis, University Medical Center Utrecht, 3Center for Experimental Medicine, Queens University, Northern Ireland, 4Hadassah Hebrew University Medical Center, Jerusalem, 5Centro Fibrosi Cistica, Azienda Ospedaliera Universitaria Integrata, 6Service de Pneumologie et Allergologie Pédiatriques and Center de Ressources et de Compétence de la Mucoviscidose, Hôpital Necker Enfants Malades, 7INSERM U 1151, Institut Necker Enfants Malades Here, we present a standardized protocol to measure the nasal potential difference (NPD). Cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) function are evaluated by the change in the voltage across the nasal epithelium after superfusion of solutions that modify ion channel activity, providing an outcome measure. Genetics Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System (REQS) Louis P. Watanabe1, Nicole C. Riddle1 1Department of Biology, University of Alabama at Birmingham The Rotating Exercise Quantification System (REQS) can induce exercise in Drosophila melanogaster through rotation while simultaneously measuring the amount of activity performed by the animals. Here, we present a point-by-point protocol detailing how to measure activity levels of animals experiencing rotational exercise treatments using the REQS. Chemistry Essential Metal Uptake in Gram-negative Bacteria: X-ray Fluorescence, Radioisotopes, and Cell Fractionation Christopher D. Radka1, Lauren L. Radford2, Adriana V.F. Massicano2, Lawrence J. DeLucas3, Suzanne E. Lapi2, Stephen G. Aller4 1Graduate Biomedical Sciences Microbiology Theme, University of Alabama at Birmingham, 2Department of Radiology, University of Alabama at Birmingham, 3Office of the Provost, University of Alabama at Birmingham, 4Department of Pharmacology and Toxicology, University of Alabama at Birmingham A protocol for the extraction of a periplasmic transition metal chaperone in the context of its native binding partners, and biophysical characterization of its substrate contents by X-ray fluorescence and radiometal uptake is presented. Neuroscience Fiber Connections of the Supplementary Motor Area Revisited: Methodology of Fiber Dissection, DTI, and Three Dimensional Documentation Baran Bozkurt1, Kaan Yagmurlu2, Erik H. Middlebrooks3, Zuzan Cayci4, Orhun M. Cevik1, Ali Karadag5, Sean Moen1, Necmettin Tanriover6, Andrew W. Grande1 1Department of Neurosurgery, University of Minnesota, 2Department of Neurosurgery, Barrow Neurological Institute, St. Josephs Hospital and Medical Center, 3Department of Radiology, University of Alabama at Birmingham, 4Department of Radiology, University of Minnesota, 5Department of Neurosurgery, Tepecik Training and Research Hospital, 6Department of Neurosurgery, Cerrahpasa Medical School, University of Istanbul The purpose of this study is to show each step of the fiber dissection technique on human cadaveric brains, the 3D documentation of these dissections, and the diffusion tensor imaging of the anatomically dissected fiber pathways. Immunology and Infection A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis Kaitlyn Schaaf1, Samuel R. Smith1, Virginia Hayley1, Olaf Kutsch1, Jim Sun1 1Department of Medicine, University of Alabama at Birmingham New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening. Developmental Biology Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair Wuqiang Zhu1, Ling Gao1, Jianyi Zhang1 1Department of Biomedical Engineering, School of Medicine, School of Engineering, University of Alabama at Birmingham We present three novel and more efficient protocols for differentiating human induced pluripotent stem cells into cardiomyocytes, endothelial cells, and smooth muscle cells and a delivery method that improves the engraftment of transplanted cells by combining cell injection with patch-mediated cytokine delivery. Biology Studying Mitochondrial Structure and Function in Drosophila Ovaries Danitra J Parker1, Aida Moran1, Kasturi Mitra1 1Department of Genetics, School of Medicine, University of Alabama at Birmingham Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper. Genetics Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System Isabelle R. Miousse1, Igor Koturbash1, Marie-Cécile Chalbot2, Martin Hauer-Jensen3, Ilias Kavouras2, Rupak Pathak3 1Department of Occupational and Environmental Health, University of Arkansas for Medical Sciences, 2Department of Environmental Health Sciences, University of Alabama at Birmingham, 3Division of Radiation Health, University of Arkansas for Medical Sciences This protocol describes techniques for the quantification and characterization of chromosomal aberrations in vitro in RAW264.7 mouse macrophages after treatment with ambient air particulate matter. Immunology and Infection Determining Immune System Suppression versus CNS Protection for Pharmacological Interventions in Autoimmune Demyelination Kirsten S. Evonuk*1, Carson E. Moseley*2, Ryan E. Doyle1, Casey T. Weaver2, Tara M. DeSilva1,3,4 1Physical Medicine and Rehabilitation, University of Alabama at Birmingham, 2Department of Pathology, University of Alabama at Birmingham, 3Department of Neurobiology, University of Alabama at Birmingham, 4Center for Glial Biology and Medicine, University of Alabama at Birmingham This protocol describes how to determine whether pharmacological treatments for experimental autoimmune encephalomyelitis show CNS protection as a consequence of suppressing immune cell infiltration or are neuroprotective during the onslaught of immune cell infiltration. Developmental Biology Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo Stefanie M. Percival1, John M. Parant1 1Department of Pharmacology and Toxicology, University of Alabama at Birmingham Mitosis is critical to every living organism and defects often lead to cancer and developmental disorders. Using this imaging protocol and zebrafish as a model system, researchers can visualize mitosis in a live vertebrate organism and the multitude of defects that arise when mitotic processes are defective. Medicine Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing Ashley N. Gilbert1, Rachael S. Shevin4, Joshua C. Anderson2, Catherine P. Langford3, Nicholas Eustace2, G. Yancey Gillespie3, Raj Singh4, Christopher D. Willey2 1Biomedical Engineering, University of Alabama at Birmingham, 2Radiation Oncology, University of Alabama at Birmingham, 3Neurosurgery, University of Alabama at Birmingham, 4Vivo Biosciences, Inc. Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis. Neuroscience Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation Hsiang-Chin Lu1, Wei-Jen Chang1, Wei-Pang Chang2, Bai-Chuang Shyu1 1Institute of Biomedical Sciences, Academia Sinica, 2Department of Anesthesiology, University of Alabama at Birmingham Studies have shown that cathodal transcranial direct-current stimulation can produce suppressive effects on drug-resistant seizures. In this study, an in vitro experimental setup was devised in which the direct-current stimulation and multielectrode array recording of seizure-like activity were evaluated in mice brain slice preparation. The direct-current stimulation parameters were evaluated. Bioengineering Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates Kayla F. Goliwas1, Lindsay M. Miller2, Lauren E. Marshall2, Joel L. Berry2, Andra R. Frost1 1Department of Pathology, University of Alabama at Birmingham, 2Department of Biomedical Engineering, University of Alabama at Birmingham We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained. Immunology and Infection Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay Alex G. Dalecki1, Cameron L. Crawford1, Frank Wolschendorf1 1Department of Medicine, Division of Infectious Diseases, University of Alabama at Birmingham Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms. Immunology and Infection Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem Xiaoyu Liu1, Yali Sun1, Camilla J Kørner1, Xinran Du1, Marie E. Vollmer1, Karolina M. Pajerowska-Mukhtar1 1Department of Biology, University of Alabama at Birmingham Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves. Biology Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1) Sinifunanya E. Nwaobi1, Michelle L. Olsen1 1Department of Cell Developmental and Integrative Biology, University of Alabama at Birmingham DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression. Immunology and Infection Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches Linlin Gu1, Anitra L. Farrow1, Alexandre Krendelchtchikov1, Qiana L. Matthews1,2 1Department of Medicine, University of Alabama at Birmingham, 2Center for AIDS Research, University of Alabama at Birmingham Here, we present a protocol to generate a proof-of-principle divalent adenovirus type 5 (Ad5) vector Ad5/H5-HVR1-KWAS-HVR5-His6 by utilizing the Antigen Capsid-Incorporation strategy. This vector was demonstrated to exhibit qualitative fitness, the capability to escape Ad5-positive sera in vitro, and the antigenicity as well as immunogenicity to the incorporated antigens. Medicine Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model Hyunki Kim1, Sharon Samuel1, John W. Totenhagen1, Marie Warren1, Jeffrey C. Sellers2, Donald J. Buchsbaum3 1Radiology, University of Alabama at Birmingham, 2Comprehensive Cancer Center, University of Alabama at Birmingham, 3Radiation Oncology, University of Alabama at Birmingham The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy. Biology Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer Jiajie Yan*1, Justin K. Thomson*1, Weiwei Zhao1, Vladimir G. Fast2, Tong Ye3, Xun Ai1 1Department of Cell and Molecular Physiology, Loyola University Chicago, 2Department of Biomedical Engineering, University of Alabama at Birmingham, 3Department of Bioengineering, Clemson University This article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers. Immunology and Infection Human Neutrophil Flow Chamber Adhesion Assay Yebin Zhou1, Dennis F. Kucik2,3,4, Alexander J. Szalai5, Jeffrey C. Edberg5 1Genetics and Genomic Sciences Graduate Program, University of Alabama at Birmingham, 2Birmingham Veterans Affairs Medical Center, 3Department of Pathology, University of Alabama at Birmingham, 4Department of Biomedical Engineering, University of Alabama at Birmingham, 5Department of Medicine, University of Alabama at Birmingham A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress. Biology Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos Shannon N. Romano1, Daniel A. Gorelick1 1Department of Pharmacology and Toxicology, University of Alabama at Birmingham Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos. Biology Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages Jacob Michael Froehlich1, Iban Seiliez2, Jean-Charles Gabillard3, Peggy R. Biga1 1Department of Biology, University of Alabama at Birmingham, 2Nutrition Métabolisme Aquaculture, INRA UR1067, 3Laboratoire de Physiologie et Genomique des Poissons, INRA UR1037 In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages. Immunology and Infection Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets Philip A. Kramer*1, Balu K. Chacko*1, Saranya Ravi1, Michelle S. Johnson1, Tanecia Mitchell1, Victor M. Darley-Usmar*1 1UAB Mitochondrial Medicine Laboratory, Center for Free Radical Biology, Department of Pathology, University of Alabama at Birmingham Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells. Immunology and Infection Assays for the Identification of Novel Antivirals against Bluetongue Virus Linlin Gu1, Stewart W. Schneller2, Qianjun Li1 1Division of Infectious Diseases, Department of Medicine, University of Alabama at Birmingham, 2Molette Laboratory for Drug Discovery, Department of Chemistry and Biochemistry, Auburn University Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals. Biology Prostaglandin Extraction and Analysis in Caenorhabditis elegans Jeevan K. Prasain1, Hieu D. Hoang2, Johnathan W. Edmonds2, Michael A. Miller2 1Department of Pharmacology and Toxicology, University of Alabama at Birmingham, 2Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS. Neuroscience Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay Somaira Nowsheen1, Fen Xia2, Eddy S. Yang1,3 1Department of Radiation Oncology, University of Alabama-Birmingham, 2Department of Radiation Oncology, The Ohio State University Medical School, 3Department of Cell Biology, and Pharmacology and Toxicology, Comprehensive Cancer Center, University of Alabama at Birmingham School of Medicine, University of Alabama-Birmingham The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage. Medicine Surgical Technique for Spinal Cord Delivery of Therapies: Demonstration of Procedure in Gottingen Minipigs Thais Federici1, Carl V. Hurtig1, Kentrell L. Burks1, Jonathan P. Riley1, Vibhor Krishna2, Brandon A. Miller1, Eric A. Sribnick1, Joseph H. Miller3, Natalia Grin1, Jason J. Lamanna1,4,5, Nicholas M. Boulis1 1Department of Neurosurgery, Emory University, 2Department of Neuroscience, Medical University of South Carolina, 3Division of Neurosurgery, University of Alabama, Birmingham, 4Department of Biomedical Engineering, Georgia Institute of Technology, 5Department of Biomedical Engineering, Emory University Short visual description of the surgical technique and device used for the delivery of (gene and cell) therapies into the spinal cord. The technique is demonstrated in the animal but is entirely translatable and currently being used for human application. Medicine Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease Lingling Guo1, Anupam Agarwal2, James F. George1 1Department of Surgery, The University of Alabama at Birmingham, 2Department of Medicine, The University of Alabama at Birmingham We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous. Neuroscience Investigating the Neural Mechanisms of Aware and Unaware Fear Memory with fMRI David C. Knight1, Kimberly H. Wood1 1Department of Psychology, University of Alabama at Birmingham A methodology to investigate the neural mechanisms that support aware and unaware memory processes during fear conditioning is described. This method monitors blood oxygen level dependent (BOLD) functional magnetic resonance imaging, skin conductance response, and unconditioned stimulus expectancy during Pavlovian fear conditioning to assess the neural correlates of distinct memory processes. Medicine Probing the Brain in Autism Using fMRI and Diffusion Tensor Imaging Rajesh K. Kana1, Donna L. Murdaugh1, Lauren E. Libero1, Mark R. Pennick1, Heather M. Wadsworth1, Rishi Deshpande1, Christi P. Hu1 1Department of Psychology, University of Alabama at Birmingham Neuroimaging techniques, such as functional MRI and Diffusion Tensor Imaging have become increasingly useful in characterizing the cognitive and neural deficits in autism. An examination of brain connectivity in autism at a network level along with adaptations for scanning children with developmental disabilities is presented. Medicine The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse Gozde Colak1, Anthony J. Filiano2, Gail V.W. Johnson3 1Department of Pharmacology and Physiology, University of Rochester, 2Department of Neurology, University of Alabama at Birmingham, 3Departments of Anesthesiology, Pharmacology and Physiology, University of Rochester Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available. Medicine Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents Amy N. Turk1, Stephanie J. Byer1, Kurt R. Zinn2, Steven L. Carroll1,3 1Department of Pathology, University of Alabama at Birmingham - UAB, 2Department of Radiology, University of Alabama at Birmingham - UAB, 3Department of Cell Biology and Neurobiology, University of Alabama at Birmingham - UAB A method for reliably grafting luciferase-tagged human malignant peripheral nerve sheath tumor cells into the sciatic nerve of immunodeficient mice is described. The use of bioluminescence imaging to demonstrate proper establishment of tumor grafts and criteria for random segregation of animals into study groups are also discussed. Biology Bioenergetic Profile Experiment using C2C12 Myoblast Cells David G. Nicholls1, Victor M. Darley-Usmar2, Min Wu3, Per Bo Jensen3, George W. Rogers3, David A. Ferrick3 1Buck Institute for Age Research, Novato, CA, 2Department of Pathology, Center for Free Radical Biology, University of Alabama at Birmingham - UAB, 3Seahorse Bioscience, North Billerica, MA A description of a method for profiling mitochondrial function in cells is provided. The mitochondrial profile generated provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity.
