University of Texas, Austin View Institution's Website 50 articles published in JoVE Genetics Daily Transfers, Archiving Populations, and Measuring Fitness in the Long-Term Evolution Experiment with Escherichia coli Jeffrey E. Barrick1,2, Zachary D. Blount2,3, Devin M. Lake2,4, Jack H. Dwenger1, Jesus E. Chavarria-Palma1, Minako Izutsu2,3, Michael J. Wiser2,5 1Department of Molecular Biosciences, The University of Texas at Austin, 2BEACON Center for the Study of Evolution in Action, Michigan State University, 3Department of Microbiology and Molecular Genetics, Michigan State University, 4Department of Integrative Biology, Michigan State University, 5Biological Sciences Program, Michigan State University This protocol describes how to maintain the Escherichia coli Long-Term Evolution Experiment (LTEE) by performing its daily transfers and periodic freeze-downs and how to conduct competition assays to measure fitness improvements in evolved bacteria. These procedures can serve as a template for researchers starting their own microbial evolution experiments. Genetics Screening Sperm for the Rapid Isolation of Germline Edits in Zebrafish Brittney Voigt1, Ryoko Minowa2, Ryan S. Gray1,2 1Department of Molecular Biosciences, The University of Texas at Austin, 2Department of Nutritional Sciences, The University of Texas at Austin CRISPR-Cas technologies have revolutionized the field of genome editing. However, finding and isolating the desired germline edit remains a major bottleneck. Therefore, this protocol describes a robust method for quickly screening F0 CRISPR-injected zebrafish sperm for germline edits using standard PCR, restriction digest, and gel electrophoresis techniques. Bioengineering In Vitro Selection of Aptamers to Differentiate Infectious from Non-Infectious Viruses Marcos Ezequiel Gramajo*1, Ryan J. Lake*2, Yi Lu3, Ana Sol Peinetti1 1INQUIMAE (CONICET), Departamento de Química Inorgánica, Analítica y Química Física, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, 2Department of Chemistry, University of Illinois at Urbana-Champaign, 3Department of Chemistry, University of Texas at Austin We provide a protocol that can be generally applied to select aptamers that bind to infectious viruses only and not to viruses that have been rendered non-infectious by a disinfection method or to any other similar viruses. This opens the possibility of determining infectivity status in portable and rapid tests. Bioengineering A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans Jyoti Dubey1,2,3,4, Sudip Mondal1,5, Sandhya P. Koushika2 1National Centre for Biological Sciences, TIFR, 2Department of Biological Sciences, TIFR, 3InSTEM, 4Manipal Academy of Higher Education, 5Department of Mechanical Engineering, The University of Texas at Austin The protocol describes a simple microfluidic chip design and microfabrication methodology used to grow C. elegans in presence of a continuous food supply for up to 36 h. The growth and imaging device also enables intermittent long-term high-resolution imaging of cellular and sub-cellular processes during development for several days. Biochemistry Modeling an Enzyme Active Site using Molecular Visualization Freeware Kristen Procko1, Sandy Bakheet1, Josh T. Beckham1, Margaret A. Franzen2, Henry Jakubowski3, Walter R. P. Novak4 1The University of Texas at Austin, 2Mount Mary University, 3College of St. Benedict/St. John’s University, 4Wabash College A key skill in biomolecular modeling is displaying and annotating active sites in proteins. This technique is demonstrated using four popular free programs for macromolecular visualization: iCn3D, Jmol, PyMOL, and UCSF ChimeraX. Biology Preparing and Rearing Axenic Insects with Tissue Cultured Seedlings for Host-Gut Microbiota Interaction Studies of the Leaf Beetle Meiqi Ma1, Pei Liu1, Jingya Yu2, Runhua Han3, Letian Xu1 1State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, 2Institute of Plant Protection, Wuhan Institute of Landscape Architecture, 3McKetta Department of Chemical Engineering, University of Texas at Austin To obtain an axenic insect, its egg surface is sterilized, and the hatched larva is subsequently reared using axenic leaves. This method provides an efficient way for axenic insect preparation without administering antibiotics or developing an artificial diet, which can also be applied to other leaf-eating insects. Biology Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series Jonathan D. Partridge1, Rasika M. Harshey1 1Department of Molecular Biosciences, The University of Texas at Austin Many bacteria use flagella-driven motility to navigate their environment and colonize favorable surroundings both individually and as a collective. Demonstrated here is the use of three established methods that exploit motility as a selection tool to identify components/pathways contributing to swimming and swarming motility. Engineering Evaluation of an Exclusive Spur Dike U-Turn Design with Radar-Collected Data and Simulation Yang Shao1, Hongtao Yu1, Huan Wu2, Xueyan Han1, Xizhen Zhou1, Christian G. Claudel3, Hualing Zhang4, Chen Yang5 1 This protocol describes the process of solving a microscopic traffic problem with simulation. The whole process contains a detailed description of data collection, data analysis, simulation model build, simulation calibration, and sensitive analysis. Modifications and troubleshooting of the method are also discussed. Immunology and Infection Use of Viral Entry Assays and Molecular Docking Analysis for the Identification of Antiviral Candidates against Coxsackievirus A16 Jonathan Y. Wang1, Chien-Ju Lin2, Ching-Hsuan Liu3,4, Liang-Tzung Lin3,5 1Department of Molecular Biosciences, University of Texas at Austin, 2School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, 3Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, 4Department of Microbiology and Immunology, Dalhousie University, 5Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University The goal of the protocol is to illustrate the different assays relating to viral entry that can be used to identify candidate viral entry inhibitors. Genetics Antibody-Free Assay for RNA Methyltransferase Activity Analysis Samantha B. Shelton1, Blerta Xhemalce1 1Department of Molecular Biosciences, University of Texas at Austin Here, an antibody-free in vitro assay for direct analysis of methyltransferase activity on synthetic or in vitro transcribed RNA is described. Medicine Preclinical Model of Hind Limb Ischemia in Diabetic Rabbits Andrew D. Sligar1, Gretchen Howe2, Julia Goldman3, Patricia Felli2, Varsha Karanam1, Richard W. Smalling2,4, Aaron B. Baker1,5,6,7 1Department of Biomedical Engineering, University of Texas at Austin, 2Division of Cardiology, University of Texas McGovern Medical School, 3Center for Laboratory Animal Medicine and Care, UT Health Science Center at Houston, 4Memorial Herman Heart and Vascular Center, Texas Medical Center, 5Institute for Cellular and Molecular Biology, University of Texas at Austin, 6The Institute for Computational Engineering and Sciences, University of Texas at Austin, 7Institute for Biomaterials, Drug Delivery and Regenerative Medicine, University of Texas at Austin We describe a surgical procedure used to induce peripheral ischemia in rabbits with hyperlipidemia and diabetes. This surgery acts as a preclinical model for conditions experienced in peripheral artery disease in patients. Angiography is also described as a means to measure the extent of introduced ischemia and recovery of perfusion. Developmental Biology An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors Cody O. Crosby1, Janet Zoldan1 1Department of Biomedical Engineering, University of Texas at Austin Endothelial progenitors derived from induced pluripotent stem cells (iPSC-EPs) have the potential to revolutionize cardiovascular disease treatments and to enable the creation of more faithful cardiovascular disease models. Herein, the encapsulation of iPSC-EPs in three-dimensional (3D) collagen microenvironments and a quantitative analysis of these cells’ vasculogenic potential are described. Neuroscience Stimulus-specific Cortical Visual Evoked Potential Morphological Patterns Julia Campbell1,2, Mashhood Nielsen1,2, Alison LaBrec1,2, Connor Bean1,2 1Department of Communication Sciences and Disorders, The University of Texas at Austin, 2Central Sensory Processes Laboratory, The University of Texas at Austin In this paper, we present a protocol to investigate differential cortical visual evoked potential morphological patterns through stimulation of ventral and dorsal networks using high-density EEG. Visual object and motion stimulus paradigms, with and without temporal jitter, are described. Visual evoked potential morphological analyses are also outlined. Immunology and Infection Quantitative PCR of T7 Bacteriophage from Biopanning Xiujuan Peng1, Jasmim Leal1, Rashmi Mohanty1, Melissa Soto1, Debadyuti Ghosh1 1Division of Molecular Pharmaceutics and Drug Delivery, College of Pharmacy, University of Texas at Austin A reproducible, accurate, and time efficient quantitative PCR (qPCR) method to enumerate T7 bacteriophage is described here. The protocol clearly describes phage genomic DNA preparation, PCR reaction preparation, qPCR cycling conditions, and qPCR data analysis. Environment The Calibration and Use of Capacitance Sensors to Monitor Stem Water Content in Trees Ashley M. Matheny1, Steven R. Garrity2, Gil Bohrer3 1Department of Geological Sciences, Jackson School of Geosciences, University of Texas at Austin, 2METER Group, Inc., USA, 3Department of Civil, Environmental and Geodetic Engineering, Ohio State University The hydraulic capacitance of biomass is a key component of the vegetation water budget, which serves as a buffer against short and long-term drought stresses. Here, we present a protocol for the calibration and use of soil moisture capacitance sensors to monitor water content in the stems of large trees. Behavior Task Interruption and Resumption Paradigm for Testing the Activation and Pursuit of an Abstract Thinking Goal Jae-Eun Namkoong1, Marlone D. Henderson2 1Managerial Sciences Department, University of Nevada, 2Department of Psychology, University of Texas This protocol was designed to test the activation and pursuit of cognitive goals (e.g., an abstract thinking goal) using the task interruption and resumption paradigm. The protocol is suitable for cognitive goals that are automatically pursued once activated, as the distraction procedure prevents goal pursuit during the interruption period. Bioengineering Intraductal Delivery to the Rabbit Mammary Gland Amelia Clark1, Nora K. Bird2, Amy Brock1 1Department of Biomedical Engineering, The University of Texas at Austin, 2Department of Anesthesiology, UTMB Health at Galveston Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents. Genetics Tools to Study the Role of Architectural Protein HMGB1 in the Processing of Helix Distorting, Site-specific DNA Interstrand Crosslinks Anirban Mukherjee1, Karen M. Vasquez1 1Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin Targeted DNA damage can be achieved by tethering a DNA damaging agent to a triplex-forming oligonucleotide (TFO). Using modified TFOs, DNA damage-specific protein association, and DNA topology modification can be studied in human cells by the utilization of modified chromatin immunoprecipitation assays and DNA supercoiling assays described herein. Medicine Ultrasound Assessment of Flow-Mediated Dilation of the Brachial and Superficial Femoral Arteries in Rats Daniel R. Machin1, Miriam E. Leary2, Yuxia He1,3, Yan-Ting Shiu1,3, Hirofumi Tanaka2, Anthony J. Donato1,4,5,6 1Department of Internal Medicine, University of Utah, 2Department of Kinesiology and Health Education, University of Texas at Austin, 3Division of Nephrology and Hypertension, University of Utah, 4Department of Biochemistry, University of Utah, 5Department of Exercise and Sport Science, University of Utah, 6Geriatric Research Education and Clinical Center, Department of Veterans Affairs Non-invasive assessment of endothelial function in humans can be determined by the flow-mediated dilation technique. Although thousands of studies have used this technique, no study has performed this technique non-invasively in rats. The following article describes non-invasive measurement of flow-mediated dilation in the brachial and superficial femoral arteries of rats. Chemistry Sulfate Separation by Selective Crystallization with a Bis-iminoguanidinium Ligand Charles A. Seipp1,2, Neil J. Williams1,3, Radu Custelcean1 1Chemical Sciences Division, Oak Ridge National Laboratory, 2Department of Chemistry, The University of Texas at Austin, 3Department of Chemistry, The University of Tennessee A protocol for in situ aqueous synthesis of a bis(iminoguanidinium) ligand and its utilization in selective separation of sulfate is presented. Bioengineering Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device Hyun Jung Kim1, Jaewon Lee1, Jin-Ha Choi1, Anthony Bahinski2, Donald E. Ingber2,3,4 1Department of Biomedical Engineering, The University of Texas at Austin, 2Wyss Institute for Biologically Inspired Engineering at Harvard University, 3 We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system. Chemistry Epitaxial Growth of Perovskite Strontium Titanate on Germanium via Atomic Layer Deposition Edward L. Lin1, Bryce I. Edmondson1, Shen Hu1, John G. Ekerdt1 1McKetta Department of Chemical Engineering, The University of Texas at Austin This work details the procedures for the growth and characterization of crystalline SrTiO3 directly on germanium substrates by atomic layer deposition. The procedure illustrates the ability of an all-chemical growth method to integrate oxides monolithically onto semiconductors for metal-oxide semiconductor devices. Engineering Electroactive Polymer Nanoparticles Exhibiting Photothermal Properties Travis Cantu1, Bradley Rodier2, Zachary Iszard2, Alissa Kilian2, Varun Pattani3, Kyle Walsh2, Katharina Weber2, James Tunnell3, Tania Betancourt1,2, Jennifer Irvin1,2 1Materials Science, Engineering, and Commercialization Program, Texas State University, 2Department of Chemistry and Biochemistry, Texas State University, 3Department of Biomedical Engineering, The University of Texas at Austin A protocol is presented for the synthesis and preparation of nanoparticles consisting of electroactive polymers. Environment A CO2 Concentration Gradient Facility for Testing CO2 Enrichment and Soil Effects on Grassland Ecosystem Function Philip A. Fay1, Lara G. Reichmann2, Michael J. Aspinwall2, Albina R. Khasanova1, H. Wayne Polley1 1USDA-ARS, Grassland Soil and Water Research Laboratory, 2Department of Integrative Biology, University of Texas at Austin The Lysimeter Carbon Dioxide Gradient Facility creates a 250 to 500 µl L-1 linear carbon dioxide gradient in temperature-controlled chambers housing grassland plant communities on clay, silty clay, and sandy soil monoliths. The facility is used to determine how past and future carbon dioxide levels affect grassland carbon cycling. Medicine Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch Victoria Le1, Collin G. Johnson1, Jonathan D. Lee1, Aaron B. Baker1 1Department of Biomedical Engineering, University of Texas at Austin We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis. Chemistry Synthesis of Immunotargeted Magneto-plasmonic Nanoclusters Chun-Hsien Wu1,2, Konstantin Sokolov1,2 1Department of Biomedical Engineering, University of Texas at Austin, 2Department of Imaging Physics, University of Texas M.D. Anderson Cancer Center Here, we describe a protocol for synthesis of magneto-plasmonic nanoparticles with a strong magnetic moment and a strong near-infrared (NIR) absorbance. The protocol also includes antibody conjugation to the nanoparticles through the Fc moiety for various biomedical applications which require molecular specific targeting. Bioengineering Lipid Bilayer Vesicle Generation Using Microfluidic Jetting Christopher W. Coyne1, Karan Patel1, Johanna Heureaux1, Jeanne Stachowiak3, Daniel A. Fletcher4,5, Allen P. Liu1,2 1Department of Mechanical Engineering, University of Michigan, 2Department of Biomedical Engineering, University of Michigan, 3Department of Biomedical Engineering, Institute for Cellular and Molecular Biology, The University of Texas at Austin, 4Department of Bioengineering, University of California, Berkeley, 5Physical Biosciences Division, Lawrence Berkeley National Laboratory Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired. Biology Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy Patrick D. McGurk1, C. Ben Lovely1, Johann K. Eberhart1 1Institute for Cell and Molecular Biology, The University of Texas at Austin Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos. Engineering Experimental Measurement of Settling Velocity of Spherical Particles in Unconfined and Confined Surfactant-based Shear Thinning Viscoelastic Fluids Sahil Malhotra1, Mukul M. Sharma1 1Petroleum & Geosystems Engineering, The University of Texas at Austin This paper demonstrates the experimental procedure to measure terminal settling velocities of spherical particles in surfactant-based shear thinning viscoelastic fluids. Fluids over a wide range of rheological properties are prepared and settling velocities are measured for a range of particle sizes in unbounded fluids and fluids between parallel walls. Behavior Assessing Forelimb Function after Unilateral Cervical SCI using Novel Tasks: Limb Step-alternation, Postural Instability and Pasta Handling Zin Z. Khaing*1, Sydney A. Geissler*1, Timothy Schallert2, Christine E. Schmidt1,3 1Department of Biomedical Engineering, The University of Texas at Austin, 2Department of Psychology, The University of Texas at Austin, 3The J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida Three new behavioral tests (forelimb step-alternation, postural instability test, pasta handling test) for evaluating forelimb function after cervical spinal cord injury in rodents are described. Chemistry Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria Jeremy C. Henderson1, John P. O'Brien2, Jennifer S. Brodbelt2, M. Stephen Trent1,3 1Section of Molecular Genetics and Microbiology, The University of Texas at Austin, 2Department of Chemistry and Biochemistry, The University of Texas at Austin, 3The Institute of Cellular and Molecular Biology, The University of Texas at Austin Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses. Immunology and Infection Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages Farshad Tamari1, Joanna Tychowski2, Laura Lorentzen3 1Department of Biological Sciences, Kingsborough Community College, 2Institute for Cellular and Molecular Biology, University of Texas at Austin, 3New Jersey Center for Science, Technology and Mathematics, Kean University We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin that did not significantly and adversely affect cell survival by solubilizing the fatty acids and the toxin and using them in cell survival assays. Immunology and Infection Mass Spectrometric Analysis of Glycosphingolipid Antigens Alexandra Bili Yin1, David Hawke2, Dapeng Zhou3,4 1Undergraduate Program, Rice University, 2Proteomics Facility, Department of Pathology, University of Texas MD Anderson Cancer Center, 3Department of Melanoma Medical Oncology, University of Texas MD Anderson Cancer Center, 4University of Texas Graduate School of Biological Sciences at Houston A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards. Immunology and Infection Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells Ivan Liadi1, Jason Roszik2, Gabrielle Romain1, Laurence J.N. Cooper2, Navin Varadarajan1 1Department of Chemical and Biomolecular Engineering, University of Houston, 2Division of Pediatrics, Research Unit 907, University of Texas MD Anderson Cancer Center We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis. Neuroscience Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography Christina J. Schier1, Regina A. Mangieri1, Geoffrey A. Dilly1, Rueben A. Gonzales1 1College of Pharmacy, Division of Pharmacology and Toxicology, The University of Texas at Austin A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated. Neuroscience Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution Gayathri Nattar Ranganathan1, Helmut J. Koester1 1Section of Neurobiology, Center for Learning and Memory, The University of Texas at Austin Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo. Bioengineering Engineering a Bilayered Hydrogel to Control ASC Differentiation Shanmugasundaram Natesan1, David O. Zamora1, Laura J. Suggs2, Robert J. Christy1 1Department of Extremity Trauma Research and Regenerative Medicine, United States Army Institute of Surgical Research, 2Department of Biomedical Engineering, The University of Texas at Austin This protocol focuses on utilizing the inherent ability of stem cells to take cue from their surrounding extracellular matrix and be induced to differentiate into multiple phenotypes. This methods manuscript extends our description and characterization of a model utilizing a bilayered hydrogel, composed of PEG-fibrin and collagen, to simultaneously co-differentiate adipose-derived stem cells1. Neuroscience High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain Sucharit Katyal1, Clint A. Greene1, David Ress1 1Psychology & Neurobiology, Imaging Research Center & Center for Perceptual Systems, The University of Texas at Austin This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example. Medicine A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice Jacqueline F. Donoghue1, Oliver Bogler2, Terrance G. Johns1 1Monash Institute of Medical Research, 2MD Anderson Cancer Centre, University of Texas In order to evaluate novel therapeutic paradigms for the treatment of glioma, physiological relevant models are essential. We utilize an implantable guide screw procedure for establishment of intracranial xenograft models that is more rapid and safer than stereotactic approaches. Neuroscience Examining Local Network Processing using Multi-contact Laminar Electrode Recording Bryan J. Hansen1, Sarah Eagleman1, Valentin Dragoi2 1Graduate School of Biomedical Science, Neuroscience Program, University of Texas, 2Department of Neurobiology and Anatomy, University of Texas A fundamental issue in our understanding of cortical circuitry is how networks in different cortical layers encode sensory information. Here we describe electrophysiological techniques utilizing multi-contact laminar electrodes to record single-units and local field potentials and present analyses to identify cortical layers. Bioengineering Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3 Jialie Luo1, Yingmin Zhu1, Michael X. Zhu1, Hongzhen Hu1 1Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center at Houston This video provides a detailed protocol for studying the pharmacological profile of human TRPA1 channels using FlexStation 3. The protocol covers details of cell preparation, dye loading and operation of the microplate reader, FlexStation 3. Immunology and Infection 'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens David A. Schofield1, Ian J. Molineux2, Caroline Westwater3 1BioSciences Division, Guild Associates, Inc., 2Department of Molecular Genetics and Microbiology, University of Texas at Austin, 3Department of Craniofacial Biology, Medical University of South Carolina A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis. Immunology and Infection Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells Srinivas S. Somanchi1, Vladimir V. Senyukov1, Cecele J. Denman1, Dean A. Lee1 1Division of Pediatrics, MD Anderson Cancer Center - University of Texas Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function. Bioengineering High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging Mark Pierce1, Dihua Yu2, Rebecca Richards-Kortum1 1Department of Bioengineering, Rice University, 2Department of Molecular and Cellular Oncology, The Univeristy of Texas M. D. Anderson Cancer Center In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies. Neuroscience How to Build a Laser Speckle Contrast Imaging (LSCI) System to Monitor Blood Flow Adrien Ponticorvo1, Andrew K. Dunn1 1Biomedical Engineering Department, University of Texas at Austin This video demonstrates how to build a Laser Speckle Contrast Imaging (LSCI) system that can easily be used to monitor blood flow. Neuroscience Novel Apparatus and Method for Drug Reinforcement Allison A. Feduccia1, Christine L. Duvauchelle1 1College of Pharmacy, Division of Pharmacology and Toxicology, University of Texas at Austin Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in research to model various components of drug reinforcement, consumption, and addiction in humans. In this report, we combined traditional CPP and self-administration methods as a novel approach to studying drug reinforcement and addiction in rats. Neuroscience Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats Esther Y. Maier1,2, Sean T. Ma3, Allison Ahrens2,4, Timothy J. Schallert2,4,5, Christine L. Duvauchelle1,2,4 1College of Pharmacy, Division of Pharmacology and Toxicology, University of Texas at Austin, 2The Waggoner Center of Addiction and Alcohol Research, University of Texas at Austin, 3Department of Psychology, University of Michigan, 4Institute for Neuroscience, University of Texas at Austin, 5Department of Psychology, University of Texas at Austin Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience. Neuroscience The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice Kelly A. Tennant1, Aaron L. Asay2, Rachel P. Allred3, Angela R. Ozburn4, Jeffrey A. Kleim5, Theresa A. Jones1,2 1Institute for Neuroscience, University of Texas at Austin, 2Department of Psychology, University of Texas at Austin, 3Department of Neurology, University of Florida, 4Department of Psychiatry, University of Texas Southwestern Medical Center, 5Department of Neuroscience, McKnight Brain Institute, University of Florida The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments. Immunology and Infection Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques Hong He1, Amy N. Courtney1, Eric Wieder2, K. Jagannadha Sastry1 1Department of Immunology, MD Anderson Cancer Center - University of Texas, 2Department of Medicine, University of Miami We demonstrate the utility of multicolor flow cytometry for detailed phenotypic and functional characterization of total as well as memory subsets of CD4+ and CD8+ T cells in rhesus macaques, the ideal model for HIV/AIDS vaccine studies. Biology Monitoring Plant Hormones During Stress Responses Marie J. Engelberth1, Jurgen Engelberth1 1Department of Biology, University of Texas A simple method is provided that allows for the rapid extraction and analysis of multiple plant hormones from small tissue samples. The procedure uses vapor phase extraction as the solemn purification step. Samples are analyzed by GC/MS with chemical ionization that produces mainly (M+1)+ ions.