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42 ARTICLES PUBLISHED IN JoVE

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Immunology and Infection

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce
Bledar Bisha 1, Byron F. Brehm-Stecher 2
1Center for Meat Safety and Quality, Department of Animal Sciences, Colorado State University, 2Rapid Microbial Detection and Control Laboratory, Department of Food Science and Human Nutrition, Iowa State University

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

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Immunology and Infection

Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection
Theodore E. Johnson *1, Brady A. Michel *1, Crystal Meyerett 1, Angela Duffy 1, Anne Avery 1, Steven Dow 1, Mark D. Zabel 1
1Department of Microbiology, Immunology and Pathology, Colorado State University

Here we describe a novel assay for monitoring prion uptake and trafficking by immune cells immediately following intraperitoneal inoculation by purifying and fluorescently labeling aggregated prion rods from infected brain material then monitoring their uptake and movement from the injection site and characterizing the cells mediating these events.

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Biology

In vivo and in vitro Studies of Adaptor-clathrin Interaction
Daniel Feliciano 1, Jarred J. Bultema 1, Andrea L. Ambrosio 1, Santiago M. Di Pietro 1
1Department of Biochemistry and Molecular Biology, Colorado State University

Clathrin-mediated endocytosis depends on adaptor proteins that coordinate cargo selection and clathrin coat assembly. Here we describe procedures to study adaptor-clathrin physical interaction and live cell imaging approaches using as a model the yeast endocytic adaptor protein Sla1p.

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Immunology and Infection

Alphavirus Transducing System: Tools for Visualizing Infection in Mosquito Vectors
Aaron Phillips 1, Eric Mossel 1, Irma Sanchez-Vargas 1, Brian Foy 1, Ken Olson 1
1Microbiology, Immunology, and Pathology, Colorado State University

Methods for using alphavirus transducing systems to express fluorescent reporters in vitro and in adult mosquitoes are described. This technique may be adapted to express any protein of interest in lieu of or in addition to a reporter.

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Immunology and Infection

Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry
Manira Rayamajhi 1, Elizabeth F. Redente 2, Tracy V. Condon 1, Mercedes Gonzalez-Juarrero 3, David W.H. Riches 1,2,4, Laurel L. Lenz 1,4
1Department of Immunology, University of Colorado School of Medicine, 2Division of Cell Biology, Department of Pediatrics, National Jewish Health , 3Department of Microbiology, Immunology, and Pathology, Colorado State University, 4Department of Immunology, National Jewish Health

We illustrate non-surgical delivery of test materials into the lungs of anesthetized mice via the trachea. This method permits lung exposure to bacterial and viral pathogens, cytokines, antibodies, beads, chemicals, or dyes. We further describe harvesting and processing of lungs and lung draining lymph nodes (LDLNs) for flow cytometry.

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Immunology and Infection

DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat (VNTR) - Fragment Length Analysis (FLA)
Ronald W. Jensen 1, Jason Rivest 1, Wei Li 1, Varalakshmi Vissa 1
1Department of Microbiology, Immunology and Pathology, Colorado State University

Leprosy, caused by Mycobacterium leprae, is still endemic in many places. In order to learn about the spread and mode of transmission of leprosy, it is important to determine which strain of M. leprae has infected a patient. Variable numbers of tandem repeats (VNTR) typing is one such method.

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Medicine

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies
Wei Li 1, Shu Zhu 1, Yusong Zhang 1, Jianhua Li 1, Andrew E. Sama 1, Ping Wang 1, Haichao Wang 1
1The Feinstein Institute for Medical Research, North Shore – LIJ Health System

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

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Behavior

P50 Sensory Gating in Infants
Anne Spencer Ross 1,2, Sharon Kay Hunter 1, Mark A Groth 1, Randal Glenn Ross 1
1Department of Psychiatry, University of Colorado School of Medicine, 2Colorado State University

Methods to record auditory P50 sensory gating, a physiological marker of cerebral inhibition which reflects early stages of attention, are described.

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Chemistry

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)
Corey D. Broeckling 1, Adam L. Heuberger 1, Jessica E. Prenni 1
1Proteomics and Metabolomics Facility, Colorado State University

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

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Medicine

Transabdominal Ultrasound for Pregnancy Diagnosis in Reeves' Muntjac Deer
Kelly D. Walton 1, Erin McNulty 1, Amy V. Nalls 1, Candace K. Mathiason 1
1Department of Microbiology, Immunology, and Pathology, Colorado State University

Transabdominal ultrasound is described as an effective, noninvasive means for assessing reproductive status in Reeves' muntjac deer. These methods can be used to achieve early pregnancy diagnosis and to evaluate fetal viability. Future applications of this technique include estimation of gestational age and effects of maternal disease on fetal development.

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Environment

High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities
Colin W. Bell 1, Barbara E. Fricks 1, Jennifer D. Rocca 1, Jessica M. Steinweg 2, Shawna K. McMahon 3, Matthew D. Wallenstein 1
1Natural Resource Ecology Laboratory, Colorado State University, 2Biosciences Division, Oak Ridge National Laboratory, 3Department of Bioengineering, University of Colorado

To measure potential rates of soil extracellular enzyme activities, synthetic substrates that are bound to a fluorescent dye are added to soil samples. Enzyme activity is measured as the fluorescent dye is released from the substrate by an enzyme-catalyzed reaction, where higher fluorescence indicates more substrate degradation.

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Biology

Milk Collection Methods for Mice and Reeves' Muntjac Deer
Kassandra Willingham 1, Erin McNulty 1, Kelly Anderson 1, Jeanette Hayes-Klug 1, Amy Nalls 1, Candace Mathiason 1
1Department of Microbiology, Immunology, and Pathology, Prion Research Center, Colorado State University

Milk collection from animal models facilitates various research avenues: understanding passive immunity, identifying pathogens responsible for vertical transmission and, through the use of transgenic mice, even commercial production of proteins found in human breast milk. Here we illustrate a simple method for milk collection in mice and Reeves’ muntjac deer.

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Environment

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling
Jennifer L Soong 1, Dan Reuss 1, Colin Pinney 1, Ty Boyack 1, Michelle L Haddix 1, Catherine E Stewart 2, M. Francesca Cotrufo 1,3
1Natural Resource Ecology Laboratory, Colorado State University, 2Soil Plant Nutrient REsearch, USDA-ARS, 3Department of Soil and Crop Sciences, Colorado State University

This method explains how to build and operate a continuous 13C and 15N isotope labeling chamber for uniform or differential plant tissue labeling. Representative results from metabolic and structural labeling of Andropogon gerardii are discussed.

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Environment

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water
Bledar Bisha 1, Jaclyn A. Adkins 2, Jana C. Jokerst 3, Jeffrey C. Chandler 1, Alma Pérez-Méndez 4, Shannon M. Coleman 4, Adrian O. Sbodio 5, Trevor V. Suslow 5, Michelle D. Danyluk 6, Charles S. Henry 2, Lawrence D. Goodridge 7
1Department of Animal Science, University of Wyoming, 2Department of Chemistry, Colorado State University, 3Department of Environmental and Radiological Health Sciences, Colorado State University, 4Department of Animal Sciences, Colorado State University, 5Department of Plant Sciences, University of California, Davis, 6Department of Food Science and Human Nutrition, Citrus Research and Education Center, University of Florida, 7Department of Food Science and Agricultural Chemistry, McGill University

A protocol involving integrated concentration, enrichment, and end-point colorimetric detection of foodborne pathogens in large volumes of agricultural water is presented here. Water is filtered through Modified Moore Swabs (MMS), enriched with selective or non-selective media, and detection is performed using paper-based analytical devices (µPAD) imbedded with bacterial-indicative colorimetric substrates.

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Environment

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability
Jodi R. Schilz 1, K. J. Reddy 2, Sreejayan Nair 3, Thomas E. Johnson 4, Ronald B. Tjalkens 5, Kem P. Krueger 3, Suzanne Clark 6
1Division of Physical Therapy, Department of Orthopedics & Rehabilitation, University of New Mexico, 2Department of Ecosystem Science and Management, University of Wyoming, 3School of Pharmacy, University of Wyoming, 4Department of Environmental and Radiological Health Sciences, Colorado State University, 5Center for Environmental Medicine, Colorado State University, 6College of Pharmacy, California Northstate University

Production bleed water (PBW) was treated with cupric oxide nanoparticles (CuO-NPs) and cellular toxicity was assessed in cultured human cells. The goal of this protocol was to integrate the native environmental sample into a cell culture format assessing the changes in toxicity due to CuO-NP treatment.

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JoVE Core

SSVEP-based Experimental Procedure for Brain-Robot Interaction with Humanoid Robots
Jing Zhao 1, Wei Li 1,2, Xiaoqian Mao 1, Mengfan Li 1
1School of Electrical Engineering and Automation, Tianjin University, 2Department of Computer & Electrical Engineering and Computer Science, California State University

The overall goal of this method is to establish an SSVEP-based experimental procedure by integrating multiple software programs to enable the study of brain-robot interaction with humanoid robots, which is prospective in assisting the sick and elderly as well as performing unsanitary or dangerous jobs.

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Bioengineering

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes
Heather R. Bender 1, Sarah Kane 1, Mark D. Zabel 1
1Department of Microbiology, Immunology, and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

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Medicine

Ferric Chloride-induced Murine Thrombosis Models
Wei Li 1,2, Marvin Nieman 3, Anirban Sen Gupta 4
1Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, 2Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, 3Department of Pharmacology, Case Western Reserve University, 4Department of Biomedical Engineering, Case Western Reserve University

We report a refined procedure of the ferric chloride (FeCl3)-induced thrombosis models on carotid and mesenteric artery as well as vein, characterized efficiently using intravital microscopy to monitor time to occlusive thrombi formation.

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Environment

Integrating Remote Sensing with Species Distribution Models; Mapping Tamarisk Invasions Using the Software for Assisted Habitat Modeling (SAHM)
Amanda M. West 1, Paul H. Evangelista 1, Catherine S. Jarnevich 2, Nicholas E. Young 1, Thomas J. Stohlgren 1, Colin Talbert 2, Marian Talbert 3, Jeffrey Morisette 3, Ryan Anderson 1
1Natural Resource Ecology Laboratory, Colorado State University, 2U.S. Geological Survey, Fort Collins Science Center, 3U.S. Geological Survey - U.S. Department of the Interior, North Central Climate Science Center

We demonstrate the utility of remotely sensed data and the newly developed Software for Assisted Habitat Modeling (SAHM) in predicting invasive species occurrence on the landscape. An ensemble of predictive models produced highly accurate maps of tamarisk (Tamarix spp.) invasion in Southeastern Colorado, USA when assessed with subsequent field validations.

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Immunology and Infection

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function
Mia J. Smith 1,2, Thomas A. Packard 1, Shannon K. O'Neill 1, Rochelle M. Hinman 1, Marynette Rihanek 3, Peter A. Gottlieb 3, John C. Cambier 1,4
1Department of Immunology and Microbiology, University of Colorado School of Medicine, 2Department of Microbiology, Immunology, and Pathology, Colorado State University, 3Barbara Davis Center for Childhood Diabetes, University of Colorado School of Medicine, 4Department of Biomedical Research, National Jewish Health

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

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Immunology and Infection

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone
James Weger-Lucarelli 1, Nisha K. Duggal 2, Aaron C. Brault 2, Brian J. Geiss 1, Gregory D. Ebel 1
1Department of Microbiology, Immunology, and Pathology, Colorado State University, 2Division of Vector-Borne Diseases, Centers for Disease Control and Prevention

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

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Neuroscience

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay
Wei Li 1,2, Chen Zhang 1, Xiulian Sun 3
1Department of Neurology, Qilu Hospital of Shandong University, 2Department of Neurology, Qingdao Municipal Hospital, 3Brain Research Institute, Qilu Hospital of Shandong University

This protocol aims to describe a method to examine the Ca2+ retention capacity and Ca2+- triggered mitochondrial swelling of isolated mitochondria of SH-SY5Y cells step-by-step.

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Neuroscience

Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System
Benjamin B. Fixman *1, Isaac W. Babcock *1, Laurie S. Minamide *1, Alisa E. Shaw 1, Marina I. Oliveira da Silva 1,2, Avery M. Runyan 1, Michael T. Maloney 1,3, Jeffrey J. Field 1, James R. Bamburg 1
1Department of Biochemistry and Molecular Biology and Molecular, Cellular and Integrated Neuroscience Program, Colorado State University, 2IBMC-Instituto de Biologia Molecular e Celular, i3S-Instituto de Investigaçãoe Inovação em Saúde, ICBAS, Universidade do Porto, 3Denali Therapeutics

Presented here is a modified roller tube method for culturing and intermittent high-resolution imaging of rodent brain slices over many weeks with precise repositioning on photoetched coverslips. Neuronal viability and slice morphology are well maintained. Applications of this fully enclosed system using viruses for cell-type specific expression are provided.

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Education

The Diffusion of Passive Tracers in Laminar Shear Flow
Manuchehr Aminian 1,2, Francesca Bernardi 1, Roberto Camassa 1, Daniel M. Harris 1,3, Richard M. McLaughlin 1
1Department of Mathematics, University of North Carolina at Chapel Hill, 2Department of Mathematics, Colorado State University, 3School of Engineering, Brown University

A protocol for the study of the diffusion of passive tracers in laminar pressure-driven flow is presented. The procedure is applicable to various capillary pipe geometries.

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Biochemistry

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum
Gustavo Diaz 1, Chandler Bridges 1, Megan Lucas 1, Yong Cheng 2, Jeff S. Schorey 2, Karen M. Dobos 1, Nicole A. Kruh-Garcia 1
1Department of Microbiology, Immunology and Pathology, Colorado State University, 2Department of Biological Sciences, Eck Institute for Global Health, University of Notre Dame

Here, we present a protocol to purify exosomes from both plasma and serum with reduced co-purification of non-exosomal blood proteins. The optimized protocol includes ultrafiltration, protease treatment, and size exclusion chromatography. Enhanced purification of exosomes benefits downstream analyses, including more accurate quantification of vesicles and proteomic characterization.

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Environment

Detection of Viruses from Bioaerosols Using Anion Exchange Resin
Joshua W. Schaeffer 1, Jeffrey C. Chandler 2, Margaret Davidson 1,3, Sheryl L. Magzamen 1, Alma Pérez-Méndez 4, Stephen J. Reynolds 1, Lawrence D. Goodridge 5, John Volckens 6, Alan B. Franklin 2, Susan A. Shriner 2, Bledar Bisha 7
1High Plains Intermountain Center for Agricultural Health and Safety, Department of Environmental and Radiological Health Sciences, Colorado State University, 2National Wildlife Research Center, Wildlife Services, Animal and Plant Health Inspection Service, United States Department of Agriculture, 3Western Sydney University, 4Leprino Foods, Inc, 5Department of Food Science and Agricultural Chemistry, McGill University, 6Department of Mechanical Engineering, Colorado State University, 7Department of Animal Science, University of Wyoming

An anion exchange resin-based method, adapted to liquid impingement-based bioaerosol sampling of viruses is demonstrated. When coupled with downstream molecular detection, the method allows for facile and sensitive detection of viruses from bioaerosols.

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Behavior

The Use of Traditional Fear Tests to Evaluate Different Emotional Circuits in Cattle
Courtney Lynd Daigle 1, Amanda J. Hubbard 1, Temple Grandin 2
1Animal Behavior & Welfare Laboratory, Department of Animal Science, Texas A&M University, 2Department of Animal Sciences, Colorado State University

Here, we present a protocol to conduct a variety of behavioral tests in cattle that have been designed to evaluate emotions. A battery of behavioral tests (open field test, startle test, bovine zero maze, exit velocity, pen score, and chute score) were conducted to evaluate different components of animal temperament.

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Chemistry

Close-Space Sublimation-Deposited Ultra-Thin CdSeTe/CdTe Solar Cells for Enhanced Short-Circuit Current Density and Photoluminescence
Alexandra M. Bothwell 1, Jennifer A. Drayton 1, Pascal M. Jundt 1, James R. Sites 1
1Department of Physics, Colorado State University

This work describes the complete fabrication process of thin absorber cadmium selenium telluride/cadmium telluride photovoltaic devices for enhanced efficiency. The process utilizes an automated in-line vacuum system for close-space sublimation deposition that is scalable, from fabrication of small area research devices as well as large-scale modules.

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Chemistry

Direct-Coupled Electroretinogram (DC-ERG) for Recording the Light-Evoked Electrical Responses of the Mouse Retinal Pigment Epithelium
Kiyoharu J. Miyagishima 1, Congxiao Zhang *2, Volha V. Malechka *3, Kapil Bharti 2, Wei Li 1
1Retinal Neurophysiology Section, National Eye Institute, National Institutes of Health, 2Ocular and Stem Cell Translational Research Section, National Eye Institute, National Institutes of Health, 3Human Visual Function Core, National Eye Institute, National Institutes of Health

Here, we present a method for recording light-evoked electrical responses of the retinal pigment epithelium (RPE) in mice using a technique known as DC-ERGs first described by Marmorstein, Peachey, and colleagues in the early 2000s.

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Behavior

Revised and Neuroimaging-Compatible Versions of the Dual Task Screen
Allan M. Aumen *1, Kelly J. Oberg *1, Susan M. Mingils 2, Cecelia B. Berkner 3, Brian L. Tracy 3, Jaclyn A. Stephens 1,2
1Molecular Cellular and Integrative Neuroscience Program, Colorado State University, 2Dept. of Occupational Therapy, Colorado State University, 3Dept. of Health and Exercise Science, Colorado State University

We developed the original Dual Task Screen (DTS) as a portable, low-cost measure that can evaluate athletes with sports-induced mild traumatic brain injury. We revised the original DTS for future clinical use and developed a neuroimaging-compatible version of the DTS to measure neural underpinnings of single and dual task performance.

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Engineering

Design and Development of a Three-Dimensionally Printed Microscope Mask Alignment Adapter for the Fabrication of Multilayer Microfluidic Devices
Celine R. Garcia *1, Zhenya Ding *1, Hilario C. Garza 1, Wei Li 1
1Department of Chemical Engineering, Texas Tech University

This project allows small laboratories to develop an easy-to-use platform for the fabrication of precise multilayer microfluidic devices. The platform consists of a three-dimensionally printed microscope mask alignment adapter using which multilayer microfluidic devices with alignment errors of <10 µm were achieved.

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Biology

Bead Loading Proteins and Nucleic Acids into Adherent Human Cells
Charlotte Ayn Cialek 1, Gabriel Galindo 1, Amanda Lynn Koch 1, Matthew Neeley Saxton 1, Timothy John Stasevich 1,2
1Department of Biochemistry and Molecular Biology, Colorado State University, 2World Research Hub Initiative, Institute of Innovative Research, Tokyo Institute of Technology

Bead loading introduces proteins, plasmids, and particles into adherent mammalian cells. This cell loading technique is inexpensive, rapid, and does not substantially affect cell health. It is best suited for live-cell imaging.

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JoVE Journal

Identification of RNA Fragments Resulting from Enzymatic Degradation using MALDI-TOF Mass Spectrometry
Shawn W. Schowe 1, Conner J. Langeberg 2, Erich G. Chapman 2, Kitty Brown 3, Marino J. E. Resendiz 1
1Department of Chemistry, University of Colorado, Denver, 2Department of Chemistry & Biochemistry, University of Denver, 3Analytical Resources Core Bioanalysis & Omics, Colorado State University

MALDI-TOF was used to characterize fragments obtained from the reactivity between oxidized RNA and the exoribonuclease Xrn-1. The present protocol describes a methodology that can be applied to other processes involving RNA and/or DNA.

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Immunology and Infection

Mycobacterium tuberculosis Extracellular Vesicle Enrichment through Size Exclusion Chromatography
Joan M. Ryan 1, Karen M. Dobos 1, Nicole A. Kruh-Garcia 1,2
1Mycobacteria Research Laboratories, Department of Microbiology Immunology & Pathology, Colorado State University, 2BioMARC, Infectious Disease Research Center, Colorado State University

This protocol describes size exclusion chromatography, a facile and reproducible technique for enriching Mycobacterium tuberculosis extracellular vesicles from culture supernatants.

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Biology

Hand Dissection of Caenorhabditis elegans Intestines
Jessica L. Hill 1, Andrew Moore 1, Robert T. P. Williams 1, Erin Osborne Nishimura 1
1Department of Biochemistry & Molecular Biology, Colorado State University

The present protocol describes a procedure for isolating intestines from adult Caenorhabditis elegans nematode worms by hand for input in genomics, proteomics, microbiome, or other assays.

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Environment

Visualizing Field Data Collection Procedures of Exposure and Biomarker Assessments for the Household Air Pollution Intervention Network Trial in India
Karthikeyan D. Rajamani 1, Sankar Sambandam 1, Krishnendu Mukhopadhyay 1, Naveen Puttaswamy 1, Gurusamy Thangavel 1, Durairaj Natesan 1, Rengaraj Ramasamy 1, Saritha Sendhil 1, Amudha Natarajan 1, Vigneswari Aravindalochan 1, Ajay Pillarisetti 2, Michael Johnson 3, Joshua Rosenthal *4, Kyle Steenland 5, Ricardo Piedhrahita 3, Jennifer Peel 6, Maggie L. Clark 6, Dana Boyd Barr 5, Sarah Rajkumar 6, Bonnie Young 6, Shirin Jabbarzadeh 7, Ghislaine Rosa 8, Miles Kirby 9, Lindsay J. Underhill 10, Anaite Diaz-Artiga 11, Amy Lovvorn 5, William Checkley 12, Thomas Clasen 5, Kalpana Balakrishnan 1
1Department of Environmental Health Engineering, ICMR Center for Advanced Research on Air Quality, Climate and Health, Faculty of Public Health, Sri Ramachandra Institute of Higher Education and Research (Deemed University), 2Division of Environmental Health Sciences, University of California, Berkeley, 3Berkeley Air Monitoring Group, 4Division of International Epidemiology and Population Studies, National Institutes of Health, 5Gangarosa Department of Environmental Health, Rollins School of Public Health, Emory University, 6Department of Environmental and Radiological Health Sciences, Colorado State University, 7Department of Biostatistics and Informatics, Rollins School of Public Health, Emory University, 8Department of Disease Control, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, 9Department of Global Health & Population, Harvard, T.H. Chan School of Public Health, 10Cardiovascular Division, Washington University School of Medicine, Washington University, 11Centro de Estudios en Salud, Universidad del Valle de Guatemala, 12Division of Pulmonary and Critical Care, School of Medicine, Johns Hopkins University

We detail the consistent, high-quality procedures used throughout air and biological sampling processes at Indian field sites during a large randomized controlled trial. Insights gathered from the oversight of applications of innovative technologies, adapted for exposure assessment in rural regions, enable better field data collection practices with more reliable outcomes.

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Biology

Primary Culture of Porcine Retinal Pigment Epithelial Cells
Feng Wen *1,2,3, Yanzi Wang *1,2,3, Danxue He 1,2,3, Chunyan Liao 1,2,3, Weijie Ouyang 1,2,3, Zuguo Liu 1,2,3, Wei Li 1,2,3, Yi Liao 1,2,3
1Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, School of Medicine, Xiamen University, 2Department of Ophthalmology, Xiang'an Hospital of Xiamen University, School of Medicine, Xiamen University, 3Xiamen University Affiliated Xiamen Eye Center, School of Medicine, Xiamen University

Here, an easy-to-follow method to culture primary porcine retinal pigment epithelial cells in vitro is presented.

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Transgenic Manipulation of Arthropod Vectors: Tools to Study Vector-Borne Diseases
Adeline E. Williams 1,2, Kenneth E. Olson 1
1Department of Microbiology, Immunology, Pathology, Colorado State University, 2Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health

Transgenic Manipulation of Arthropod Vectors: Tools to Study Vector-Borne Diseases

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Biology

Quantifying Fitness Costs in Transgenic Aedes aegypti Mosquitoes
Adeline E. Williams 1,2, Irma Sanchez-Vargas 1, Lindsay E. Martin 2,3, Ines Martin-Martin 2,4, Susi Bennett 1, Ken E. Olson 1, Eric Calvo 2
1Center for Vector-borne Infectious Diseases, Department of Microbiology, Immunology, and Pathology, Colorado State University, 2Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 3Department of Biological Sciences, Vanderbilt University, 4National Center for Microbiology, Instituto de Salud Carlos III

The present protocol describes how to measure common life parameter data in Aedes aegypti mosquitoes, including fecundity, wing size, fertility, sex ratio, viability, development times, male contribution, and adult longevity. These measurements can be used to assess the fitness of transgenic mosquitoes.

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Neuroscience

Free-Hand Intracerebroventricular Injections in Mice
Richard B. McCosh 1, Lauren A. Young 1
1Department of Biomedical Sciences, Colorado State University

Here, a simple and rapid approach for performing intracerebroventricular injections in mice using a free-hand approach (that is, without a stereotaxic device) is described.

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Neuroscience

Adipose-Derived Mesenchymal Stromal Cells Co-Cultured with Primary Mixed Glia to Reduce Prion-Induced Inflammation
Arielle J. D. Hay 1,2, Katriana A. Popichak 1,2, Mark D. Zabel 1,2, Julie A. Moreno 1,3
1Prion Research Center, Colorado State University, 2Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, 3Department of Environmental and Radiological Health Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University

Adipose-derived mesenchymal stromal cells (AdMSCs) have potent immunomodulatory properties useful for treating diseases associated with inflammation. We demonstrate how to isolate and culture murine AdMSCs and primary mixed glia, stimulate AdMSCs to upregulate anti-inflammatory genes and growth factors, assess migration of AdMSCs, and co-culture AdMSCs with primary mixed prion-infected glia.

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JoVE Core

Disposable Dosators Intended for Dry Powder Delivery to Mice
Sara E. Maloney 1, Ian E. Stewart 1, Jeffrey B. Mecham 1, Mercedes Gonzalez-Juarrero 2, Bernd Meibohm 3, Anthony J. Hickey 1
1Technology Advancement and Commercialization, RTI International, 2Mycobacteria Research Laboratories, Department of Microbiology, Immunology, and Pathology, Colorado State University, 3Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center

Pharmaceutical dry powder development necessitates reliable in vivo testing, often using a murine model. Device technology for accurately and reproducibly delivering dry powder aerosols to mice is restricted. This study presents disposable dosators for pulmonary drug delivery at mouse-relevant doses, aiding initial proof-of-concept research.

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