University of Alabama, Birmingham

57 articles published in JoVE

• Biology

  
  Isolation of Retinal Pigment Epithelial Cells from Guinea Pig Eyes So Goto^1,2,3, Michael Frost⁴, Christine Wildsoet^{1 1}Herbert 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 Mitchell^{1 1}Department 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. Mattheyses^{1 1}Department 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 Liu^1,2, Justin D. Anderson^1,2, Jennifer Natt^1,2, Jennifer S. Guimbellot^{1,2 1}Gregory 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. Brosius^2,3, Steven L. Carroll^{1 1}Department 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. Master^{1 1}Department 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 Mitchell^{1 1}Department 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.

• Behavior

  
  Fear Incubation Using an Extended Fear-Conditioning Protocol for Rats César Acevedo-Triana^1,6, Javier L. Rico², Leonardo A. Ortega², Melissa Andrea N. Cardenas³, Fernando P. Cardenas³, Manuel J. Rojas⁴, Juan Carlos Forigua-Vargas², Julián Cifuentes², Camilo Hurtado-Parrado^{2,5 1}School 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. Edmonds^{1 1}Department 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 Bolding^{1 1}Department 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 Wajapeyee^{1 1}Department 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 Mou^{1 1}Shenzhen 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. Peters^{1 1}Cell, 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 Ubil^{2 1}Lineberger 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. Swift^1,2, Rafael Jaimes III^1,2, Damon McCullough^1,2, Morgan Burke^1,2, Marissa Reilly^1,2, Takuya Maeda^1,2,3, Hanyu Zhang⁴, Nobuyuki Ishibashi^1,2,3, Jack M. Rogers⁴, Nikki Gillum Posnack^{1,2,5 1}Center 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 Zhao^1,2,3, Chengjiang Zhao⁴, David K.C. Cooper⁵, Ying Lu², Kewang Luo⁶, Huiyun Wang³, Pengfei Chen³, Changchun Zeng³, Shaodong Luan¹, Lisha Mou², Hanchao Gao^{1,2,3 1}Department 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. Ernst^1,2, Asher Kahn-Krell¹, Chengming Fan^1,3, Danielle Pretorius¹, Hanxi Zhu¹, Xi Lou¹, Lufang Zhou², Jianyi Zhang¹, Wuqiang Zhu^{1 1}Department 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. Miller^{1 1}Department 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. Duffy^1,3, Christopher J. Kemp¹, Kelvin C. Luk⁴, Laura A. Volpicelli-Daley⁵, Nicholas M. Kanaan¹, Caryl E. Sortwell^{1,3,6 1}Department 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 Moran^1,2, Yichen Guo^3,4, Rong Yuan^1,3, Nicholas Barnekow¹, Jordan Palmer², Adam Beck³, Bin Ren^{3,4,5 1}Blood 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. Nahm^{2 1}Department 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 Kannappan^{1 1}Department 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. Gray^{1 1}Department 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 Mou^{1 1}Shenzhen 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-Gaudelus^{6,7 1}Department 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. Riddle^{1 1}Department 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. Aller^{4 1}Graduate 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. Grande^{1 1}Department 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 Sun^{1 1}Department 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 Zhang^{1 1}Department 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 Mitra^{1 1}Department 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 Pathak^{3 1}Department 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. DeSilva^{1,3,4 1}Physical 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. Parant^{1 1}Department 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. Willey^{2 1}Biomedical 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 Shyu^{1 1}Institute 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. Frost^{1 1}Department 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 Wolschendorf^{1 1}Department 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-Mukhtar^{1 1}Department 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. Olsen^{1 1}Department 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. Matthews^{1,2 1}Department 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. Buchsbaum^{3 1}Radiology, 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 Ai^{1 1}Department 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. Kucik^2,3,4, Alexander J. Szalai⁵, Jeffrey C. Edberg^{5 1}Genetics 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. Gorelick^{1 1}Department 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. Biga^{1 1}Department 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 1}UAB 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 Li^{1 1}Division 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. Miller^{2 1}Department 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. Yang^{1,3 1}Department 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. Lamanna^1,4,5, Nicholas M. Boulis^{1 1}Department 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. George^{1 1}Department 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. Wood^{1 1}Department 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. Hu^{1 1}Department 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. Johnson^{3 1}Department 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. Carroll^{1,3 1}Department 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. Ferrick^{3 1}Buck 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.