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University of Alabama at Birmingham

60 ARTICLES PUBLISHED IN JoVE

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Biology

Bioenergetic Profile Experiment using C2C12 Myoblast Cells
David G. Nicholls 1, Victor M. Darley-Usmar 2, Min Wu 3, Per Bo Jensen 3, George W. Rogers 3, David A. Ferrick 3
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.

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Medicine

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse
Gozde Colak 1, Anthony J. Filiano 2, Gail V.W. Johnson 3
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.

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Neuroscience

Investigating the Neural Mechanisms of Aware and Unaware Fear Memory with fMRI
David C. Knight 1, Kimberly H. Wood 1
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.

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Medicine

Probing the Brain in Autism Using fMRI and Diffusion Tensor Imaging
Rajesh K. Kana 1, Donna L. Murdaugh 1, Lauren E. Libero 1, Mark R. Pennick 1, Heather M. Wadsworth 1, Rishi Deshpande 1, Christi P. Hu 1
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.

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Biology

Prostaglandin Extraction and Analysis in Caenorhabditis elegans
Jeevan K. Prasain 1, Hieu D. Hoang 2, Johnathan W. Edmonds 2, Michael A. Miller 2
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.

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Immunology and Infection

Assays for the Identification of Novel Antivirals against Bluetongue Virus
Linlin Gu 1, Stewart W. Schneller 2, Qianjun Li 1
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.

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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 Ravi 1, Michelle S. Johnson 1, Tanecia Mitchell 1, 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.

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Biology

Imaging Plasma Membrane Deformations With pTIRFM
Daniel R. Passmore 1, Tejeshwar C. Rao 1, Andrew R. Peleman 1, Arun Anantharam 1
1Department of Biological Sciences, Wayne State University

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

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Biology

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages
Jacob Michael Froehlich 1, Iban Seiliez 2, Jean-Charles Gabillard 3, Peggy R. Biga 1
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.

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Immunology and Infection

Human Neutrophil Flow Chamber Adhesion Assay
Yebin Zhou 1, Dennis F. Kucik 2,3,4, Alexander J. Szalai 5, Jeffrey C. Edberg 5
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.

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Biology

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos
Shannon N. Romano 1, Daniel A. Gorelick 1
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.

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Biology

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)
Sinifunanya E. Nwaobi 1, Michelle L. Olsen 1
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.

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Biology

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer
Jiajie Yan *1, Justin K. Thomson *1, Weiwei Zhao 1, Vladimir G. Fast 2, Tong Ye 3, Xun Ai 1
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.

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Medicine

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model
Hyunki Kim 1, Sharon Samuel 1, John W. Totenhagen 1, Marie Warren 1, Jeffrey C. Sellers 2, Donald J. Buchsbaum 3
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.

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Immunology and Infection

Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches
Linlin Gu 1, Anitra L. Farrow 1, Alexandre Krendelchtchikov 1, Qiana L. Matthews 1,2
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.

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JoVE Journal

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem
Xiaoyu Liu 1, Yali Sun 1, Camilla J Kørner 1, Xinran Du 1, Marie E. Vollmer 1, Karolina M. Pajerowska-Mukhtar 1
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.

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Neuroscience

Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation
Hsiang-Chin Lu 1, Wei-Jen Chang 1, Wei-Pang Chang 2, Bai-Chuang Shyu 1
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.

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Immunology and Infection

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay
Alex G. Dalecki 1, Cameron L. Crawford 1, Frank Wolschendorf 1
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.

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Bioengineering

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates
Kayla F. Goliwas 1, Lindsay M. Miller 2, Lauren E. Marshall 2, Joel L. Berry 2, Andra R. Frost 1
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.

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Medicine

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing
Ashley N. Gilbert 1, Rachael S. Shevin 4, Joshua C. Anderson 2, Catherine P. Langford 3, Nicholas Eustace 2, G. Yancey Gillespie 3, Raj Singh 4, Christopher D. Willey 2
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.

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Research

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo
Stefanie M. Percival 1, John M. Parant 1
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.

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JoVE Journal

Determining Immune System Suppression versus CNS Protection for Pharmacological Interventions in Autoimmune Demyelination
Kirsten S. Evonuk *1, Carson E. Moseley *2, Ryan E. Doyle 1, Casey T. Weaver 2, Tara M. DeSilva 1,3,4
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.

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Genetics

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System
Isabelle R. Miousse 1, Igor Koturbash 1, Marie-Cécile Chalbot 2, Martin Hauer-Jensen 3, Ilias Kavouras 2, Rupak Pathak 3
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.

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Education

Studying Mitochondrial Structure and Function in Drosophila Ovaries
Danitra J Parker 1, Aida Moran 1, Kasturi Mitra 1
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.

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Developmental Biology

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair
Wuqiang Zhu 1, Ling Gao 1, Jianyi Zhang 1
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.

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Immunology and Infection

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis
Kaitlyn Schaaf 1, Samuel R. Smith 1, Virginia Hayley 1, Olaf Kutsch 1, Jim Sun 1
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.

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Neuroscience

Fiber Connections of the Supplementary Motor Area Revisited: Methodology of Fiber Dissection, DTI, and Three Dimensional Documentation
Baran Bozkurt 1, Kaan Yagmurlu 2, Erik H. Middlebrooks 3, Zuzan Cayci 4, Orhun M. Cevik 1, Ali Karadag 5, Sean Moen 1, Necmettin Tanriover 6, Andrew W. Grande 1
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.

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Bioengineering

A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer
Alessandra Merenda 1,2, Amanda Andersson-Rolf 1,2, Roxana C. Mustata 1, Taibo Li 1, Hyunki Kim 3, Bon-Kyoung Koo 1,2
1Wellcome Trust - Medical Research Council Stem Cell Institute, University of Cambridge, 2Department of Genetics, University of Cambridge, 3Department of Pathology, Yonsei University College of Medicine

This protocol describes the steps for cloning multiple single guide RNAs into one guide RNA concatemer vector, which is of particular use in creating multi-gene knockouts using CRISPR/Cas9 technology. The generation of double knockouts in intestinal organoids is shown as a possible application of this method.

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Medicine

Standardized Measurement of Nasal Membrane Transepithelial Potential Difference (NPD)
George M. Solomon 1, Inez Bronsveld 2, Kathryn Hayes 3, Michael Wilschanski 4, Paola Melotti 5, Steven M. Rowe 1, Isabelle Sermet-Gaudelus 6,7
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.

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Biology

A Method for Islet Transplantation to the Omentum in Mouse
Ying Lu *1, Shangyou Zou *1, Suzanne Bertera *2, Rita Bottino 2, David K.C. Cooper 3, Zhengzhao Liu 1, Yi Huang 1, Chengjun Wang 1, Chungu Hong 1, Tian He 1, Hancheng Zhang 1, Qin Huo 4, Hongxing Fu 5, Zhiming Cai 1, Lisha Mou 1
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.

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Chemistry

Essential Metal Uptake in Gram-negative Bacteria: X-ray Fluorescence, Radioisotopes, and Cell Fractionation
Christopher D. Radka 1, Lauren L. Radford 2, Adriana V.F. Massicano 2, Lawrence J. DeLucas 3, Suzanne E. Lapi 2, Stephen G. Aller 4
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.

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Genetics

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System (REQS)
Louis P. Watanabe 1, Nicole C. Riddle 1
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.

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Immunology and Infection

Assaying for Inorganic Polyphosphate in Bacteria
Arya Pokhrel 1, Jordan C. Lingo 2, Frank Wolschendorf 2, Michael J. Gray 1
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.

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Genetics

Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy
Jessica M. Miller *1, Nikhil Maneesh Mardhekar *1, Vasanthi Rajasekaran 1, Jianyi Zhang 1, Ram Kannappan 1
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.

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Immunology and Infection

A High-throughput Shigella-specific Bactericidal Assay
Hailey P. Weerts 1, Jigui Yu 2, Robert W. Kaminski 1, Moon H. Nahm 2
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.

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Biochemistry

Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity
Meng Zhao 1, Yawen Tang 1, Patrick J. Ernst 1,2, Asher Kahn-Krell 1, Chengming Fan 1,3, Danielle Pretorius 1, Hanxi Zhu 1, Xi Lou 1, Lufang Zhou 2, Jianyi Zhang 1, Wuqiang Zhu 1
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.

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Biology

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs
Yanli Zhao 1,2,3, Chengjiang Zhao 4, David K.C. Cooper 5, Ying Lu 2, Kewang Luo 6, Huiyun Wang 3, Pengfei Chen 3, Changchun Zeng 3, Shaodong Luan 1, Lisha Mou 2, Hanchao Gao 1,2,3
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, 6People's Hospital of Longhua

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.

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Neuroscience

Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo
Joseph R. Patterson 1, Nicole K. Polinski 2, Megan F. Duffy 1,3, Christopher J. Kemp 1, Kelvin C. Luk 4, Laura A. Volpicelli-Daley 5, Nicholas M. Kanaan 1, Caryl E. Sortwell 1,3,6
1Department of Translational Science and Molecular Medicine, Michigan State University, 2The Michael J. Fox Foundation for Parkinson's Research, 3Neuroscience Program, Michigan State University, 4Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, 5Center for Neurodegeneration and Experimental Therapeutics, University of Alabama at Birmingham, 6Mercy Health Hauenstein Neuroscience Medical Center

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.

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Developmental Biology

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract
Muhan Hu 1, Shara Legg 1, Michael A. Miller 1
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.

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Immunology and Infection

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model
Howard W. Davidson 1, Joseph Ray Cepeda 2, Nitin S. Sekhar 2, Junying Han 2, Ling Gao 3, Tomasz Sosinowski 1, Li Zhang 2
1Barbara Davis Center for Childhood Diabetes, University of Colorado Denver, 2Department of Medicine, Endocrinology, Diabetes & Metabolism, Baylor College of Medicine, 3Scientific Center, Shandong Provincial Hospital affiliated to Shandong University

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

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Cancer Research

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports
Kelly Pittman 1, Shelton Earp 1, Eric Ubil 2
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.

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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 Zhang 4, Nobuyuki Ishibashi 1,2,3, Jack M. Rogers 4, Nikki Gillum Posnack 1,2,5
1Sheikh Zayed Institute for Pediatric Surgical Innovation, Children's National Hospital, 2Children's National Heart Institute, Children's National Hospital, 3Center 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.

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Behavior

Fear Incubation Using an Extended Fear-Conditioning Protocol for Rats
César Acevedo-Triana 1,6, Javier L. Rico 2, Leonardo A. Ortega 2, Melissa Andrea N. Cardenas 3, Fernando P. Cardenas 3, Manuel J. Rojas 4, Juan Carlos Forigua-Vargas 2, Julián Cifuentes 2, Camilo Hurtado-Parrado 2,5
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.

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Cancer Research

Perfusion and Inflation of the Mouse Lung for Tumor Histology
Mackenzie L. Davenport 1, Taylor P. Sherrill 2, Timothy S. Blackwell 2, Mick D. Edmonds 1
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.

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Medicine

Inguinal Subcutaneous White Adipose Tissue (ISWAT) Transplantation Model of Murine Islets
Yuanzheng Peng *1, Zhicheng Zou *1, Jiao Chen 1, Hancheng Zhang 1, Ying Lu 1, Rito Bittino 2, Hongxing Fu 3, David K. C. Cooper 4, Shan Lin 1, Mengtao Cao 1, Yifan Dai 5, Zhiming Cai 1, Lisha Mou 1
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.

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Medicine

Measurement of Natural Killer Cell-Mediated Cytotoxicity and Migration in the Context of Hepatic Tumor Cells
Suresh Chava 1, Suresh Bugide 1, Romi Gupta 1, Narendra Wajapeyee 1
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.

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Developmental Biology

A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals
Courtney Barkley 1, Rosa Serra 1, Sarah B. Peters 1
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.

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Neuroscience

A Benchtop Approach to the Location Specific Blood Brain Barrier Opening using Focused Ultrasound in a Rat Model
Megan Rich 1, Quentin Whitsitt 1, Farah Lubin 2, Mark Bolding 1
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.

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Cancer Research

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone
Blake E. Hildreth III 1, Charlotte Palmer 2, Matthew J. Allen 2
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.

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Chemistry

Applying Dynamic Strain on Thin Oxide Films Immobilized on a Pseudoelastic Nickel-Titanium Alloy
Hanyu Zhang 1, Eric E. Benson 1, Kurt M. Van Allsburg 2, Elisa M. Miller 1, Drazenka Svedruzic 3
1Chemical and Nanoscience Center, National Renewable Energy Laboratory, 2Catalytic Carbon Transformation and Scale-Up Center, National Renewable Energy Laboratory, 3Biosciences Center, National Renewable Energy Laboratory

Dynamic, tensile strain is applied on TiO2 thin films to study the effects of strain on electrocatalysis, specifically proton reduction and water oxidation. TiO2 films are prepared by thermal treatment of the pseudo-elastic NiTi alloy (Nitinol).

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Medicine

Segmentation and Linear Measurement for Body Composition Analysis using Slice-O-Matic and Horos
Sean Steele 1, Fangyi Lin 1, Thien-Linh Le 1, Alexandra Medline 1, Michelle Higgins 1, Alex Sandberg 1, Sean Evans 1, Gordon Hong 2, Milton A. Williams 3, Mehmet A. Bilen 4, Sarah Psutka 5, Kenneth Ogan 1, Viraj A. Master 1
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.

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Medicine

Estimation of Urinary Nanocrystals in Humans using Calcium Fluorophore Labeling and Nanoparticle Tracking Analysis
Parveen Kumar 1, Andrew Bell 1, Tanecia Mitchell 1
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.

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Biology

Tension Gauge Tether Probes for Quantifying Growth Factor Mediated Integrin Mechanics and Adhesion
Tejeshwar C. Rao 1, Alexa L. Mattheyses 1
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.

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Medicine

Cox-Maze IV Procedure Concomitant with Valvular Surgery In Situs Inversus Dextrocardia: A Single-Center Experience in China
Cheng Luo *1, Chengming Fan *1, Hao Zhang 1, Long Song 1, Yuhong Liu 1, Liming Liu 1
1Department of Cardiovascular Surgery, the Second Xiangya Hospital, Central South University

We summarize the Cox-Maze IV procedure concomitant with valvular surgery performed in patients with situs inversus dextrocardia at this institution.

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Medicine

Ultrasound-Guided Induced Pluripotent Stem Cell-Derived Cardiomyocyte Implantation in Myocardial Infarcted Mice
Xun Wu 1, Kele Qin 1, Di Wang 2, Kun Xiang 1, Jun Peng 2, Jianjun Guo 3, Jinfu Yang 1,2, Chengming Fan 1,2,3
1Department of Cardiovascular Surgery, Second Xiangya Hospital, Central South University, 2Hunan Provincial Key Laboratory of Cardiovascular Research, 3Hunan Fangsheng Pharmaceutical Co., Ltd.

Ultrasound-guided cell delivery around the site of myocardial infarction in mice is a safe, effective, and convenient way of cell transplantation.

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Novel Translational Approaches To Study Kidney Disease
Emma Laurence 1, Parveen Kumar 1, Tanecia Mitchell 1
1Department of Urology, University of Alabama at Birmingham

Novel Translational Approaches To Study Kidney Disease

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Biology

Isolation of Retinal Pigment Epithelial Cells from Guinea Pig Eyes
So Goto 1,2,3, Michael Frost 4, Christine Wildsoet 1
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.

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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. Holz 1, Kenneth R. Sloan 2, Thomas Ach 1
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.

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Medicine

Radiation Planning Assistant - A Web-based Tool to Support High-quality Radiotherapy in Clinics with Limited Resources
Laurence Edward Court 1, Ajay Aggarwal 2, Hester Burger 3, Carlos Cardenas 4, Christine Chung 1, Raphael Douglas 1, Monique du Toit 5, Anuja Jhingran 1, Raymond Mumme 1, Sikudhani Muya 6, Komeela Naidoo 5, Jerry Ndumbalo 6, Tucker Netherton 1, Callistus Nguyen 1, Adenike Olanrewaju 1, Jeannette Parkes 3, Willie Shaw 7, Christoph Trauernicht 5, Melody Xu 8, Jinzhong Yang 1, Lifei Zhang 1, Hannah Simonds 9, Beth M. Beadle 10
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.

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Medicine

Isolation and Cryopreservation of Highly Viable Human Peripheral Blood Mononuclear Cells From Whole Blood: A Guide for Beginners
Brian Dinh 1, Marten A. Hoeksema 2, Nathanael J. Spann 3, Jacob Rendler 1, Isidoro Cobo 4,5, Christopher K. Glass 3,6, Calvin Yeang 1
1Division of Cardiology, Department of Medicine, University of California San Diego, 2Department of Medical Biochemistry, Amsterdam Immunity and Infection: Inflammatory diseases; Amsterdam Cardiovascular Sciences: Atherosclerosis and Ischemic Syndrome, Amsterdam UMC location University of Amsterdam, 3Department of Cellular and Molecular Medicine, University of California San Diego, 4Division of Clinical Immunology & Rheumatology, Department of Medicine, Heersink School of Medicine, University of Alabama at Birmingham, 5Comprehensive Arthritis, Musculoskeletal, Bone and Autoimmunity Center, Heersink School of Medicine, University of Alabama at Birmingham, 6Department of Medicine, University of California San Diego

This protocol presents an accessible guide for collecting, storing, and thawing peripheral blood mononuclear cells suitable for downstream analyses and workflows like flow cytometry and RNA sequencing. Plasma and buffy coat collections are also demonstrated.

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