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Washington State University

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16 articles published in JoVE

Immunology and Infection

Separation of Rat Epidermis and Dermis with Thermolysin to Detect Site-Specific Inflammatory mRNA and Protein Vikramsingh Gujar*¹, Michael B. Anderson*¹, Kenneth E. Miller*¹, Radhika D. Pande¹, Pranav Nawani², Subhas Das¹ ¹Department of Anatomy and Cell Biology, Oklahoma State University Center for Health Sciences, ²Institute for Shock Physics, Washington State University Presented here is a protocol for the separation of epidermis from dermis to evaluate inflammatory mediator production. Following inflammation, rat hind paw epidermis is separated from the dermis by thermolysin at 4 °C. The epidermis is then used for mRNA analysis by RT-PCR and protein evaluation by western blot and immunohistochemistry.
Biochemistry

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription Phillip Wibisono¹, Jingru Sun¹ ¹Department of Translational Medicine and Physiology, Elson S. Floyd College of Medicine, Washington State University Here, we describe a detailed protocol for isolating active nuclear extract from larval stage 4 C. elegans and visualizing transcription activity in an in vitro system.
Biochemistry

Resin-Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation Matthew J. Gaffrey¹, Nicholas J. Day¹, Xiaolu Li², Wei-Jun Qian¹ ¹Integrative Omics, Biological Sciences Division, Pacific Northwest National Laboratory, ²Bioproducts Sciences and Engineering Laboratory, Department of Biological Systems Engineering, Washington State University Protein thiol oxidation has significant implications under normal physiological and pathophysiological conditions. We describe the details of a quantitative redox proteomics method, which utilizes resin-assisted capture, isobaric labeling, and mass spectrometry, enabling site-specific identification and quantification of reversibly oxidized cysteine residues of proteins.
Chemistry

High-Temperature and High-Pressure In situ Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy Nicholas R. Jaegers¹, Wenda Hu², Yong Wang¹, Jian Zhi Hu¹ ¹Pacific Northwest National Laboratory, ²Washington State University The molecular structures and dynamics of solids, liquids, gases, and mixtures are of critical interest to diverse scientific fields. High-temperature, high-pressure in situ MAS NMR enables detection of the chemical environment of constituents in mixed phase systems under tightly controlled chemical environments.
Immunology and Infection

Studying Cryptosporidium Infection in 3D Tissue-derived Human Organoid Culture Systems by Microinjection Devanjali Dutta¹, Inha Heo^1,3, Roberta O'Connor² ¹Hubrecht Institute, Oncode Institute, Royal Netherlands Academy of Arts and Sciences (KNAW), UMC Utrecht, ²Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, ³Janssen Pharmaceutica We describe protocols to prepare oocysts and purify sporozoites for studying infection of human intestinal and airway organoids by Cryptosporidium parvum. We demonstrate the procedures for microinjection of parasites into the intestinal organoid lumen and immunostaining of organoids. Finally, we describe the isolation of generated oocysts from the organoids.
Environment

High-throughput Siderophore Screening from Environmental Samples: Plant Tissues, Bulk Soils, and Rhizosphere Soils Ricky W. Lewis*¹, Anjuman A. Islam*¹, Christine Jade Dilla-Ermita², Scot H. Hulbert², Tarah S. Sullivan¹ ¹Department of Crop and Soil Sciences, Washington State University, ²Department of Plant Pathology, Washington State University We present a protocol for rapid screening of environmental samples for siderophore potential contributing to micronutrient bioavailability and turnover in terrestrial systems.
Immunology and Infection

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis Aoi Hiroyasu¹, David C. DeWitt², Alan G. Goodman¹ ¹School of Molecular Biosciences, College of Veterinary Medicine, Washington State University, ²Integrative Physiology and Neuroscience, College of Veterinary Medicine, Washington State University This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.
Behavior

Tickling, a Technique for Inducing Positive Affect When Handling Rats Sylvie Cloutier^1,2, Megan R. LaFollette³, Brianna N. Gaskill³, Jaak Panksepp¹, Ruth C. Newberry⁴ ¹Center for the Study of Animal Well-being, Department of Integrative Physiology and Neuroscience, Washington State University, ²Canadian Council on Animal Care, ³Department of Animal Sciences, Center for Animal Welfare Science, College of Agriculture, Purdue University, ⁴Department of Animal and Aquacultural Sciences, Faculty of Biosciences, Norwegian University of Life Sciences This article demonstrates the standardized application of playful handling, a tickling technique designed to mimic rat rough-and-tumble play. This technique is effective at reducing fearful reactions to humans and generating positive affect when rats are handled for common husbandry activities and medical and research procedures such as injection.
Environment

On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System Joseph B. DeShields¹, Rachel A. Bomberger¹, James W. Woodhall², David L. Wheeler¹, Natalia Moroz¹, Dennis A. Johnson¹, Kiwamu Tanaka¹ ¹Department of Plant Pathology, Washington State University, ²Parma Research and Extension Center, University of Idaho Rapid and accurate detection of plant pathogens on-site, especially soil-borne pathogens, is crucial to prevent further inoculum production and proliferation of plant diseases in the field. The method developed here using a portable real-time PCR detection system enables on-site diagnosis under field conditions.
Developmental Biology

Collection of Post-mating Semen from the Female Reproductive Tract and Measurement of Semen Liquefaction in Mice Shuai Li¹, Wipawee Winuthayanon¹ ¹School of Molecular Biosciences, Center for Reproductive Biology, College of Veterinary Medicine, Washington State University Semen liquefaction is required for sperm to be liberated from the seminal gel. This study provides the procedures for collecting semen from the female reproductive tract post-coitus, and measuring semen liquefaction time.
Developmental Biology

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin Lucia Salz^1,2, Ryan R. Driskell^1,2 ¹ This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.
Immunology and Infection

Electroporation of Functional Bacterial Effectors into Mammalian Cells Ryan L. Sontag¹, Cosmin Mihai², Galya Orr², Alexei Savchenko³, Tatiana Skarina³, Hong Cui³, John R. Cort¹, Joshua N. Adkins¹, Roslyn N. Brown⁴ ¹Biological Sciences Division, Pacific Northwest National Laboratory, ²Environmental Molecular Science Laboratory, Pacific Northwest National Laboratory, ³Structural Proteomics Group, Ontario Center for Structural Proteomics, University of Toronto, ⁴Center for Bioproducts and Bioenergy, Washington State University Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.
Biology

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans Marshall L. Deline^1,2, Tracy L. Vrablik^1,2, Jennifer L. Watts^1,2 ¹School of Molecular Biosciences, Washington State University, ²Center for Reproductive Biology, Washington State University Caenorhabditis elegans is a useful model to explore the functions of polyunsaturated fatty acids in development and physiology. This protocol describes an efficient method of supplementing the C. elegans diet with polyunsaturated fatty acids.
Biology

Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture Graham Bailes¹, Margaret Lind¹, Andrew Ely¹, Marianne Powell², Jennifer Moore-Kucera³, Carol Miles², Debra Inglis², Marion Brodhagen¹ ¹Biology Department, Western Washington University, ²Washington State University Northwestern Research and Extension Center, ³Department of Plant and Soil Science, Texas Tech University Plastic films labeled "biodegradable" are commercially available for agricultural use as mulches. Tillage represents an attractive disposal method, but degradation under field conditions is poorly understood. The purpose of this study was to develop methods for isolating native soil fungi and bacteria that colonize plastic mulch films after field burial.
Neuroscience

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex William C. Clegern¹, Michele E. Moore¹, Michelle A. Schmidt¹, Jonathan Wisor¹ ¹Department of Veterinary & Comparative Anatomy, Pharmacology and Physiology, Sleep and Performance Research Center, WWAMI Medical Education Program, Washington State University A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.
Neuroscience

Preparation of an Awake Mouse for Recording Neural Responses and Injecting Tracers Michael A. Muniak^1,2, Zachary M. Mayko³, David K. Ryugo^2,4, Christine V. Portfors³ ¹Department of Neuroscience, Johns Hopkins University, ²Garvan Institute of Medical Research, ³School of Biological Sciences, Washington State University, ⁴Department of Otolaryngology-HNS, Johns Hopkins University Electrophysiological characterization of neuronal responses is important for understanding brain function and for guiding the placement of dyes for pathway tracing. However, many studies are performed in anesthetized animals. To understand brain function without anesthetics, we developed a method to record neuronal response properties and inject dyes in awake mouse.

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