Michigan State University

56 articles published in JoVE

• Genetics

  
  Daily Transfers, Archiving Populations, and Measuring Fitness in the Long-Term Evolution Experiment with Escherichia coli Jeffrey E. Barrick^1,2, Zachary D. Blount^2,3, Devin M. Lake^2,4, Jack H. Dwenger¹, Jesus E. Chavarria-Palma¹, Minako Izutsu^2,3, Michael J. Wiser^{2,5 1}Department 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.

• Cancer Research

  
  Co-Culture and Transduction of Murine Thymocytes on Delta-Like 4-Expressing Stromal Cells to Study Oncogenes in T-Cell Leukemia Gisele O. L. Rodrigues¹, WenQing Li¹, Sarah D. Cramer^1,2,3, Hila Y. Winer¹, Tu Chun Hsu^1,2,3, Timothy Gower⁴, Julie A. Hixon¹, Scott K. Durum^{1 1}Cytokines and Immunity Section, Cancer Innovation Laboratory, National Cancer Institute, National Institutes of Health, ²Comparative Biomedical Scientist Training Program, NIH, ³Department of Pathobiology and Diagnostic Investigation, Veterinary Diagnostic Laboratory, Michigan State University, ⁴NCI-Frederick Laboratory Animal Sciences Program This protocol describes the isolation of double-negative thymocytes from the mouse thymus followed by retroviral transduction and co-culture on the delta-like 4-expressing bone marrow stromal cell line co-culture system (OP9-DL4) for further functional analysis.

• Cancer Research

  
  X-Ray Visualization of Intraductal Ethanol-Based Ablative Treatment for Prevention of Breast Cancer in Rat Models Elizabeth Kenyon^1,2, Erin Zaluzec^1,3, Katherine Powell^1,2, Maximilian Volk^1,4, Shatadru Chakravarty^2,5, Jeremy Hix^2,6, Matti Kiupel⁷, Erik M. Shapiro², Lorenzo F. Sempere^{1,2 1}Precision Health Program, Michigan State University, ²Department of Radiology, Michigan State University, ³Department of Pharmacology & Toxicology, Michigan State University, ⁴College of Osteopathic Medicine, Michigan State University, ⁵TechInsights Inc., ⁶IQ Advanced Molecular Imaging Facility, Michigan State University, ⁷Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Michigan State University A procedure for the delivery of a chemical ablative solution to the rat mammary ductal tree for image-guided preventive treatment of breast cancer is described. Mammary epithelial cells can be targeted with minimal collateral tissue damage through cannulation directly into the nipple opening and intraductal infusion of a 70% ethanol-based ablative solution.

• Biology

  
  Plastoglobule Lipid Droplet Isolation from Plant Leaf Tissue and Cyanobacteria Kiran-Kumar Shivaiah^1,2, Febri A. Susanto^1,2, Elsinraju Devadasu^1,2, Peter K. Lundquist^{1,2 1}Department of Biochemistry and Molecular Biology, Michigan State University, ²Plant Resilience Institute, Michigan State University A fast and efficient protocol is presented for the isolation of plastoglobule lipid droplets associated with various photosynthetic organisms. The successful preparation of isolated plastoglobules is a crucial first step that precedes detailed molecular investigations such as proteomic and lipidomic analyses.

• Cancer Research

  
  Intraductal Delivery and X-ray Visualization of Ethanol-Based Ablative Solution for Prevention and Local Treatment of Breast Cancer in Mouse Models Elizabeth Kenyon^1,2, Erin K. Zaluzec^1,3, Katherine Powell^1,2, Maximilian Volk^1,4, Shatadru Chakravarty^2,5, Jeremy Hix^2,6, Ripla Arora^7,8, Jennifer J. Westerhuis^9,10, Matti Kiupel¹¹, Erik M. Shapiro^2,6, Lorenzo F. Sempere^{1,2 1}Precision Health Program, Michigan State University, ²Department of Radiology, College of Human Medicine, Michigan State University, ³Department of Pharmacology & Toxicology, College of Veterinary Medicine, Michigan State University, ⁴College of Osteopathic Medicine, Michigan State University, ⁵Advanced Materials Characterization Laboratory/Materials Research Center, Missouri University of Science and Technology, ⁶Institute for Quantitative (IQ) Health Science and Engineering Advanced Molecular Imaging Facility, Michigan State University, ⁷Department of Obstetrics Gynecology and Reproductive Biology, College of Human Medicine, Michigan State University, ⁸Institute for Quantitative (IQ) Health Science and Engineering, Michigan State University, ⁹Van Andel Research Institute, ¹⁰Miltenyi Biotec, ¹¹Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Michigan State University A method of intraductal injection of reagents for an ethanol-based ablative solution to the mouse mammary ductal tree for in vivo imaging and breast cancer prevention is described. Injection directly into the nipple opening allows for targeting mammary epithelial cells with minimal collateral tissue damage.

• Neuroscience

  
  Induction and Assessment of Levodopa-induced Dyskinesias in a Rat Model of Parkinson's Disease Margaret E. Caulfield¹, Jennifer A. Stancati¹, Kathy Steece-Collier^{1,2 1}Department of Translational Neuroscience, College of Human Medicine, Michigan State University, ²Hauenstein Neuroscience Center, Mercy Health Saint Mary's This article describes methods to induce and evaluate levodopa-induced dyskinesias in a rat model of Parkinson's disease. The protocol offers detailed information regarding the intensity and frequency of a range of dyskinetic behaviors, both dystonic and hyperkinetic, providing a reliable tool to test treatments targeting this unmet medical need.

• Bioengineering

  
  Establishing an Octopus Ecosystem for Biomedical and Bioengineering Research Tyler VanBuren^1,2, Carolina Cywiak^1,2, Petra Telgkamp^1,2, Christiane L. Mallett^3,4, Galit Pelled^{1,2,4 1}Department of Biomedical Engineering, Michigan State University, ²Neuroengineering division, The Institute for Quantitative Health Science and Engineering, Michigan State University, ³Biomedical Imaging division, The Institute for Quantitative Health Science and Engineering, Michigan State University, ⁴Department of Radiology, Michigan State University Understanding the unique physiological and anatomical structures of octopuses can greatly impact biomedical research. This guide demonstrates how to set-up and maintain a marine environment to accommodate this species and includes state-of-the-art imaging and analytical approaches to visualize octopus' nervous system anatomy and function.

• Biochemistry

  
  Generating Self-Assembling Human Heart Organoids Derived from Pluripotent Stem Cells Yonatan R. Lewis-Israeli^1,2, Brett D. Volmert^1,2, Mitchell A. Gabalski^1,2, Amanda R. Huang^1,2, Aitor Aguirre^{1,2 1}Institute for Quantitative Health Science and Engineering, Division of Developmental and Stem Cell Biology, Michigan State University, ²Department of Biomedical Engineering, College of Engineering, Michigan State University Here, we describe a protocol to create developmentally relevant human heart organoids (hHOs) efficiently using human pluripotent stem cells by self-organization. The protocol relies on the sequential activation of developmental cues and produces highly complex, functionally relevant human heart tissues.

• Biochemistry

  
  Complementation of Splicing Activity by a Galectin-3 - U1 snRNP Complex on Beads Patricia G. Voss¹, Kevin C. Haudek¹, Ronald J. Patterson², John L. Wang^{1 1}Department of Biochemistry and Molecular Biology, Michigan State University, ²Department of Microbiology and Molecular Genetics, Michigan State University This article describes the experimental procedures for (a) depletion of U1 snRNP from nuclear extracts, with concomitant loss of splicing activity; and (b) reconstitution of splicing activity in the U1-depleted extract by galectin-3 - U1 snRNP particles bound to beads covalently coupled with anti-galectin-3 antibodies.

• Medicine

  
  Setup and Execution of the Rapid Cycle Deliberate Practice Death Notification Curriculum Patrick G. Hughes¹, Kate E. Hughes², Mary J. Hughes^3,4, Lindsay Weaver⁴, Lauren E. Falvo⁴, Anna M. Bona⁴, Dylan Cooper⁴, Cherri Hobgood⁴, Rami A. Ahmed^{4 1}Florida Atlantic University Schmidt College of Medicine, ²Department of Emergency Medicine, University of Arizona College of Medicine, ³Department of Osteopathic Medical Specialties, College of Osteopathic Medicine, Michigan State University, ⁴Indiana University School of Medicine The goal is to demonstrate how to apply the rapid cycle deliberate practice debriefing technique to the GRIEV_ING death notification curriculum.

• Medicine

  
  Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model Patrick Haubruck^1,2, Aimee C. Colbath^1,3, Yolanda Liu¹, Shihani Stoner¹, Cindy Shu¹, Christopher B. Little^{1 1}Raymond Purves Bone and Joint Research Laboratory, Institute of Bone and Joint Research, Kolling Institute, Royal North Shore Hospital, University of Sydney, ²HTRG - Heidelberg Trauma Research Group, Center for Orthopedics, Trauma Surgery and Spinal Cord Injury, Trauma and Reconstructive Surgery, Heidelberg University Hospital, ³Department of Large Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

• Environment

  
  Ammonia Fiber Expansion (AFEX) Pretreatment of Lignocellulosic Biomass Shishir P. S. Chundawat¹, Ramendra K. Pal¹, Chao Zhao¹, Timothy Campbell², Farzaneh Teymouri², Josh Videto², Chandra Nielson², Bradley Wieferich³, Leonardo Sousa³, Bruce E. Dale³, Venkatesh Balan⁴, Sarvada Chipkar⁵, Jacob Aguado⁵, Emily Burke⁵, Rebecca G. Ong^{5 1}Department of Chemical and Biochemical Engineering, Rutgers-State University of New Jersey, ²Michigan Biotechnology Institute (MBI), ³Department of Chemical Engineering and Materials Science, Michigan State University, ⁴Engineering Technology Department, Biotechnology Program, College of Technology, University of Houston, ⁵Department of Chemical Engineering, Michigan Technological University Ammonia fiber expansion (AFEX) is a thermochemical pretreatment technology that can convert lignocellulosic biomass (e.g., corn stover, rice straw, and sugarcane bagasse) into a highly digestible feedstock for both biofuels and animal feed applications. Here, we describe a laboratory-scale method for conducting AFEX pretreatment on lignocellulosic biomass.

• Developmental Biology

  
  Analysis of Congenital Heart Defects in Mouse Embryos Using Qualitative and Quantitative Histological Methods Kristen Ball^1,2, Renee Kinne², Aitor Aguirre^{1,2 1}Department of Biomedical Engineering, Michigan State University, ²Institute for Quantitative Health Sciences and Engineering, Michigan State University In this protocol, we describe procedures to qualitatively and quantitatively analyze developmental phenotypes in mice associated with congenital heart defects.

• Biology

  
  Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish Savvas J. Constantinou¹, Linh Nguyen², Frank Kirschbaum², Vielka L. Salazar³, Jason R. Gallant^{1 1}Department of Integrative Biology, Michigan State University, ²Faculty of Life Sciences, Unit of Biology and Ecology of Fishes, Humboldt University, ³Department of Biology, Cape Breton University Here, a protocol is presented to produce and rear CRISPR/Cas9 genome knockout electric fish. Outlined in detail are the required molecular biology, breeding, and husbandry requirements for both a gymnotiform and a mormyrid, and injection techniques to produce Cas9-induced indel F0 larvae.

• Chemistry

  
  Enzymatic Synthesis of Epoxidized Metabolites of Docosahexaenoic, Eicosapentaenoic, and Arachidonic Acids Joseph W. Woodman¹, Maris A. Cinelli¹, Amy Scharmen-Burgdolf¹, Kin Sing Stephen Lee^{1 1}Department of Pharmacology and Toxicology, Michigan State University We present a method useful for large-scale enzymatic synthesis and purification of specific enantiomers and regioisomers of epoxides of arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) with the use of a bacterial cytochrome P450 enzyme (BM3).

• Neuroscience

  
  Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo Joseph R. Patterson¹, Nicole K. Polinski², Megan F. Duffy^1,3, Christopher J. Kemp¹, Kelvin C. Luk⁴, Laura A. Volpicelli-Daley⁵, Nicholas M. Kanaan¹, Caryl E. Sortwell^{1,3,6 1}Department of Translational Science and Molecular Medicine, Michigan State University, ² The goal of this article is to outline the steps required for the generation of fibrils from monomeric alpha-synuclein, subsequent quality control, and use of the preformed fibrils in vivo.

• Biochemistry

  
  Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids Phillip C. Delekta¹, Todd A. Lydic², Neal D. Hammer^{1 1}Department of Microbiology and Molecular Genetics, Michigan State University, ²Department of Physiology, Michigan State University This method provides a framework for studying incorporation of exogenous fatty acids from complex host sources into bacterial membranes, particularly Staphylococcus aureus. To achieve this, protocols for the enrichment of lipoprotein particles from chicken egg yolk and subsequent fatty acid profiling of bacterial phospholipids utilizing mass spectrometry are described.

• Environment

  
  Separation of Spinach Thylakoid Protein Complexes by Native Green Gel Electrophoresis and Band Characterization using Time-Correlated Single Photon Counting Eliezer Schwarz¹, Gary Blanchard^{2 1}Department of Plant Biology, Michigan State University, ²Department of Chemistry, Michigan State University Here we present a protocol to separate solubilized thylakoid complexes by Native Green Gel electrophoresis. Green gel bands are subsequently characterized by Time Correlated Single Photon Counting (TCSPC) and basic steps for data analysis are provided.

• Engineering

  
  A Silicon-tipped Fiber-optic Sensing Platform with High Resolution and Fast Response Guigen Liu*¹, Qiwen Sheng*¹, Weilin Hou², Matthew L. Reinke³, Ming Han^{1,4 1}Department of Electrical and Computer Engineering, Michigan State University, ²Naval Research Laboratory, Stennis Space Center, ³Oak Ridge National Laboratory, ⁴Department of Electrical and Computer Engineering, University of Nebraska-Lincoln This work reports an innovative silicon-tipped fiber-optic sensing platform (Si-FOSP) for high-resolution and fast-response measurement of a variety of physical parameters, such as temperature, flow, and radiation. Applications of this Si-FOSP span from oceanographic research, mechanical industry, to fusion energy research.

• Chemistry

  
  Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry Elijah N. McCool*¹, Rachele Lubeckyj*¹, Xiaojing Shen¹, Qiang Kou², Xiaowen Liu^2,3, Liangliang Sun^{1 1}Department of Chemistry, Michigan State University, ²Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, ³Center for Computational Biology and Bioinformatics, Indiana University School of Medicine A detailed protocol is described for the separation, identification, and characterization of proteoforms in protein samples using capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS). The protocol can be used for the high-resolution characterization of proteoforms in simple protein samples and the large-scale identification of proteoforms in complex proteome samples.

• Biology

  
  Determination of Reproductive Competence by Confirming Pubertal Onset and Performing a Fertility Assay in Mice and Rats Hanne M. Hoffmann^{1 1}Department of Animal Science, Michigan State University Many treatments and genetic mutations impact the timing of sexual maturity and fertility. This protocol describes a non-invasive method to evaluate pubertal onset in mice and rats prior to setting up a fertility study in sexually mature animals.

• Medicine

  
  Fracture Apparatus Design and Protocol Optimization for Closed-stabilized Fractures in Rodents Robert L Zondervan^1,2, Mitch Vorce³, Nick Servadio⁴, Kurt D. Hankenson^{2 1}College of Osteopathic Medicine, Michigan State University, ²Department of Orthopaedic Surgery, University of Michigan Medical School, ³Lymann Briggs College, Michigan State University, ⁴College of Engineering, Michigan State University The goal of the protocol is to optimize the fracture generation parameters to yield consistent fractures. This protocol accounts for the variations in bone size and morphology that may exist between animals. Additionally, a cost-effective, adjustable fracture apparatus is described.

• Biochemistry

  
  The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation Anne M. Scott¹, Ke Li¹, Weiming Li^{1 1}Department of Fisheries and Wildlife, Michigan State University Here, we present a protocol to isolate and characterize the structure, olfactory potency, and behavioral response of putative pheromone compounds of sea lampreys.

• Chemistry

  
  Characterizing Electron Transport through Living Biofilms Matthew Yates¹, Sarah Strycharz-Glaven¹, Joel Golden¹, Jared Roy^1,2, Stanislav Tsoi³, Jeffrey Erickson¹, Mohamed El-Naggar⁴, Scott Calabrese Barton⁵, Leonard Tender^{1 1}Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, ²George Mason University, ³Chemistry Division, Naval Research Laboratory, ⁴Departments of Physics, Biological Sciences, and Chemistry, University of Southern California, ⁵Department of Chemical Engineering and Materials Science, Michigan State University A protocol for measuring electrical conductivity of living microbial biofilms under physiologically relevant conditions is presented.

• Bioengineering

  
  Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties Youngmin Seo*¹, Chanhwi Park*², Jaewoo Son², Kyungwoo Lee², Jangsun Hwang², Yeonho Jo², Dohyun Lee², Muhammad Saad Khan², Sachin Ganpat Chavan², Yonghyun Choi², Dasom Kim², Assaf A Gilad³, Jonghoon Choi^{2 1}Center for Biomaterials, Biomedical Research Institute, Korea Institute of Science and Technology, ²School of Integrative Engineering, Chung-Ang University, ³Division of Synthetic Biology and Regenerative Medicine, Institute for Quantitative Health Science and Engineering, Michigan State University In this study, antimicrobial nanomaterials were synthesized by acidic oxidation of multiwalled carbon nanotubes and subsequent reductive deposition of silver nanoparticles. Antimicrobial activity and cytotoxicity tests were performed with the as-prepared nanomaterials.

• Immunology and Infection

  
  Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model Alessandra M. Agostinho Hunt¹, Jacob A. Gibson¹, Casandra L. Larrivee¹, Sandra O'Reilly², Svetlana Navitskaya², Julia V. Busik², Christopher M. Waters^{1 1}Department of Microbiology and Molecular Genetics, Michigan State University, ²Department of Physiology, Michigan State University Here we describe a novel diabetic murine model utilizing hairless mice for real-time, non-invasive, monitoring of biofilm wound infections of bioluminescent Pseudomonas aeruginosa. This method can be adapted to evaluate infection of other bacterial species and genetically modified microorganisms, including multi-species biofilms, and test the efficacy of antibiofilm strategies.

• Environment

  
  Building Double-decker Traps for Early Detection of Emerald Ash Borer Deborah G. McCullough¹, Therese M. Poland^{2 1}Department of Entomology and Department of Forestry, Michigan State University, ²Northern Research Station, USDA Forest Service Effective traps to attract and capture the emerald ash borer (EAB) are a key element of detecting and managing this invasive pest. Double-decker traps, placed in full sun near ash trees, incorporate visual and olfactory cues and were more likely to capture EAB than other trap designs in field trials.

• Behavior

  
  How to Build a Dichoptic Presentation System That Includes an Eye Tracker Cheng S. Qian¹, Jan W. Brascamp^{1,2 1}Department of Psychology, Michigan State University, ²Neuroscience Program, Michigan State University We recently proposed a method that allows dichoptic visual stimulus presentation and binocular eye tracking simultaneously¹. The key is the combination of an infrared eye tracker and the corresponding infrared-transparent mirrors. This manuscript provides an in depth protocol for initial setup and everyday operation.

• Developmental Biology

  
  Expansion and Adipogenesis Induction of Adipocyte Progenitors from Perivascular Adipose Tissue Isolated by Magnetic Activated Cell Sorting Kyan Thelen¹, Nadia Ayala-Lopez², Stephanie W. Watts², G. Andres Contreras^{1 1}Department of Large Animal Clinical Sciences, Michigan State University, ²Department of Pharmacology and Toxicology, Michigan State University Here we report a method for isolation of Adipocyte Progenitor Cell (APC) populations from Perivascular Adipose Tissue (PVAT) using Magnetic-activated Cell Sorting (MCS). This method allows for an increased isolation of APC per gram of adipose tissue when compared to Fluorescence-Activated Cell Sorting (FACS).

• Biochemistry

  
  Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples Tyler Rhinesmith¹, Bryan A. Killinger², Akhil Sharma³, Anna Moszczynska^{3 1}Physiology, Michigan State University, ²Center for Neurodegenerative Science Van Andel Institute, ³Pharmaceutical Sciences, Wayne State University A method for stabilizing and separating native protein complexes from unmodified tissue lysate using an amine-reactive protein cross-linker coupled to a novel two-dimensional polyacrylamide gel electrophoresis (PAGE) system is presented.

• Biology

  
  Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products Brad L. Upham¹, Iva Sovadinová², Pavel Babica^{2 1}Department of Pediatrics & Human Development, Institute for Integrative Toxicology, Michigan State University, ²RECETOX — Research Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

• Bioengineering

  
  Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol Patricia J. Slininger¹, Maureen A. Shea-Andersh¹, Stephanie R. Thompson¹, Bruce S. Dien¹, Cletus P. Kurtzman², Leonardo Da Costa Sousa³, Venkatesh Balan^{3 1}Bioenergy Research Unit, National Center for Agricultural Utilization Research, ²Mycotoxin Prevention and Applied Microbiology Research Unit, National Center for Agricultural Utilization Research, ³Chemical Engineering and Material Science, Great Lakes Bioenergy Center, Michigan State University Adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of Scheffersomyces stipitis strain NRRL Y-7124 that are able to rapidly consume hexose and pentose mixed sugars in enzyme saccharified undetoxified hydrolyzates and to accumulate over 40 g/L ethanol.

• Neuroscience

  
  An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons Chun Liu¹, Christina Chan^{1,2 1}Department of Chemical Engineering and Materials Science, Michigan State University, ²Department of Biochemistry and Molecular Biology, Michigan State University This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

• Biology

  
  Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat Carmen Hinojosa-Laborde¹, Brian Jespersen², Robert Shade^{3 1}Tactical Combat Casualty Care Research, U.S. Army Institute of Surgical Research, ²Department of Pharmacology and Toxicology, Michigan State University, ³Southwest National Primate Research Center, Texas Biomedical Research Institute The purpose of this protocol is to demonstrate the principles and techniques for measuring and calculating glomerular filtration rate, urine flow rate, and excretion of sodium and potassium in a rat. This demonstration can be used to provide students with an overall conceptual understanding of how to measure renal function.

• Immunology and Infection

  
  Isolation of Leukocytes from the Human Maternal-fetal Interface Yi Xu¹, Olesya Plazyo¹, Roberto Romero^1,2,3,4, Sonia S. Hassan^1,5, Nardhy Gomez-Lopez^{1,5,6 1}Perinatology Research Branch, NICHD/NIH/DHHS, ²Department of Obstetrics and Gynecology, University of Michigan, ³Department of Epidemiology and Biostatistics, Michigan State University, ⁴Department of Molecular Obstetrics and Genetics, Wayne State University, ⁵Department of Obstetrics and Gynecology, Wayne State University School of Medicine, ⁶Department of Immunology and Microbiology, Wayne State University School of Medicine Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

• Neuroscience

  
  In Situ Ca²⁺ Imaging of the Enteric Nervous System David E. Fried¹, Brian D. Gulbransen^{1 1}Department of Physiology, Michigan State University The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca²⁺ imaging.

• Biology

  
  Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research Brian Jespersen¹, Nathan R. Tykocki², Stephanie W. Watts¹, Peter J. Cobbett^{1 1}Department of Pharmacology and Toxicology, Michigan State University, ²Department of Pharmacology, University of Vermont College of Medicine This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.

• Biology

  
  In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein Dexin Sui*¹, Mengyu Liu*¹, Min-Hao Kuo^{1 1}Department of Biochemistry and Molecular Biology, Michigan State University Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer’s disease.

• Bioengineering

  
  Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis Sang-Hyuck Park¹, Rebecca Garlock Ong², Chuansheng Mei³, Mariam Sticklen^{4 1}The School of Plant Sciences, University of Arizona, ²Department of Chemical Engineering and Materials Science, DOE Great Lakes Bioenergy Research Center, Michigan State University, ³The Institute for Sustainable and Renewable Resources, The Institute for Advanced Learning and Research, ⁴Department of Plant, Soil and Microbial Sciences, Michigan State University A double stranded RNA interference (dsRNAi) technique is employed to down-regulate the maize cinnamoyl coenzyme A reductase (ZmCCR1) gene to lower plant lignin content. Lignin down-regulation from the cell wall is visualized by microscopic analyses and quantified by the Klason method. Compositional changes in hemicellulose and crystalline cellulose are analyzed.

• Biology

  
  A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey (Petromyzon marinus) Yu-Wen Chung-Davidson¹, Peter J. Davidson¹, Anne M. Scott¹, Erin J. Walaszczyk¹, Cory O. Brant¹, Tyler Buchinger¹, Nicholas S. Johnson², Weiming Li^{1 1}Department of Fisheries & Wildlife, Michigan State University, ²Hammond Bay Biological Station, Great Lakes Science Center, U.S. Geological Survey Sea lamprey lose the gall bladder and bile ducts during metamorphosis, a process similar to human biliary atresia. A new fixation and clarification method (CLARITY) was modified to visualize the entire biliary tree using laser scanning confocal microscopy. This method provides a powerful tool to study biliary degeneration.

• Behavior

  
  A Fully Automated Rodent Conditioning Protocol for Sensorimotor Integration and Cognitive Control Experiments Ali Mohebi¹, Karim G. Oweiss^{1,2,3 1}Electrical and Computer Engineering, Michigan State University, ²Neuroscience Program, Michigan State University, ³Cognitive Science Program, Michigan State University A fully automated protocol for rodent operant conditioning is proposed. The protocol relies on precise temporal control of behavioral events to investigate the extent to which this control influences neural activity underlying sensorimotor integration and cognitive control experiments.

• Biology

  
  Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method Olena Tetyuk¹, Urs F. Benning¹, Susanne Hoffmann-Benning^{1 1}Biochemistry and Molecular Biology, Michigan State Universtiy Knowledge of the composition of the phloem sap as well as the mechanism of its loading and long-distance transport is essential for the understanding of long-distance signaling in plant development and stress/pathogen response and of assimilate transport. This manuscript describes the collection of phloem exudates using the EDTA-facilitated method.

• Engineering

  
  Scanning-probe Single-electron Capacitance Spectroscopy Kathleen A. Walsh¹, Megan E. Romanowich¹, Morewell Gasseller^1,2, Irma Kuljanishvili^1,3, Raymond Ashoori⁴, Stuart Tessmer^{1 1}Department of Physics and Astronomy, Michigan State University, ²Department of Chemistry & Biochemistry/Physics, Mercyhurst University, ³Department of Physics, Saint Louis University, ⁴Department of Physics, Massachusetts Institute of Technology Scanning-probe single-electron capacitance spectroscopy facilitates the study of single-electron motion in localized subsurface regions. A sensitive charge-detection circuit is incorporated into a cryogenic scanning probe microscope to investigate small systems of dopant atoms beneath the surface of semiconductor samples.

• Biology

  
  Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity Giovanni Stefano¹, Luciana Renna¹, Federica Brandizzi^{1 1}DOE Plant Research Laboratory, Michigan State University A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.

• Bioengineering

  
  Microfluidic Mixers for Studying Protein Folding Steven A. Waldauer¹, Ling Wu¹, Shuhuai Yao², Olgica Bakajin³, Lisa J. Lapidus^{1 1}Department of Physics and Astronomy, Michigan State University, ²Department of Mechanical Engineering, Hong Kong University of Science and Technology, ³Center for Biophotonics, University of California, Davis In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 μs. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

• Biology

  
  Sexual Development and Ascospore Discharge in Fusarium graminearum Brad Cavinder¹, Usha Sikhakolli², Kayla M. Fellows³, Frances Trail^{2,4 1}Genetics Program, Michigan State University, ²Department of Plant Biology, Michigan State University, ³Human Biology Program, Michigan State University, ⁴Department of Plant Pathology, Michigan State University Sexual crosses and isolation of recombinant progeny are important research tools for the filamentous fungus, Fusarium graminearum, The techniques necessary successfully carry out these processes are presented.

• Medicine

  
  Femoral Arterial and Venous Catheterization for Blood Sampling, Drug Administration and Conscious Blood Pressure and Heart Rate Measurements Brian Jespersen¹, Lauren Knupp¹, Carrie A. Northcott^{1 1}Department of Pharmacology and Toxicology, Michigan State University Chronic catheterization of blood vessels in the rat is often required for administration of substances, obtain blood sample over a period of time or for direct conscious blood pressure measurements. Femoral arterial catheterization of the rat and corresponding measurements of blood pressure in the conscious animal will be demonstrated.

• Bioengineering

  
  GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization Jonathan Walton^1,2, Goutami Banerjee², Suzana Car^{2 1}DOE Plant Research Laboratory, Michigan State University, ²DOE Great Lakes Bioenergy Research Center, Michigan State University GENPLAT (GLBRC Enzyme Platform) is an automated platform for discovery and optimization of enzyme cocktails for biomass degradation. It can be adapted to multiple feedstocks and mixtures of enzymes containing multiple components.

• Biology

  
  Experimental Manipulation of Body Size to Estimate Morphological Scaling Relationships in Drosophila R. Craig Stillwell¹, Ian Dworkin², Alexander W. Shingleton², W. Anthony Frankino^{1 1}Department of Biology & Biochemistry, University of Houston, ²Department of Zoology, Michigan State University Morphological scaling relationships capture and describe organismal shape. We present a method to measure morphological scaling relationships across the natural range of body sizes in fully metamorphic insects. Using a simple diet manipulation we increase the distribution of trait sizes, permitting the accurate description of how shape and size co-vary.

• Neuroscience

  
  Mechanical Manipulation of Neurons to Control Axonal Development Phillip Lamoureux¹, Steven Heidemann¹, Kyle E. Miller^{1 1}Department of Zoology, Michigan State University, East Lansing Application and direct measurements of forces on neurons in the 2-1000 microdyne range are achieved with high precision using calibrated glass needles. This methodology can be used to control and measure several aspects of axonal development, including axonal initiation, axonal tension, velocity of axonal elongation, and force vectors.

• Biology

  
  Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC) Zhen Wang¹, Christoph Benning^{1 1}Department of Biochemistry and Molecular Biology, Michigan State University Composition of polar lipid extracts and the fatty acid composition of individual glycerolipids are determined in a simple and robust lipid profiling experiment. For this purpose, glycerolipids are isolated by thin layer chromatography and subjected to transmethylation of their acyl groups. Fatty acyl methylesters are quantified by gas-liquid chromatography.

• Biology

  
  Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana Sankalpi N. Warnasooriya¹, Beronda L. Montgomery^{1,2 1}Department of Energy - Plant Research Laboratory, Michigan State University (MSU), ²Department of Biochemistry and Molecular Biology, Michigan State University (MSU) The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

• Biology

  
  Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part II: Carbohydrates Cliff E. Foster¹, Tina M. Martin¹, Markus Pauly^{2 1}Great Lakes Bioenergy Research Center, Michigan State University (MSU), ²Great Lakes Bioenergy Research Center and DOE-Plant Research Lab, Michigan State University (MSU) Plant biomass is a major carbon-neutral renewable resource that could be used for the production of biofuels. Plant biomass consists mainly of cell walls, a structurally complex composite material termed lignocellulosics. Here we describe a protocol for a comprehensive analysis of the content and composition of wall derived carbohydrates.

• Biology

  
  Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part I: Lignin Cliff E. Foster¹, Tina M. Martin¹, Markus Pauly^{2 1}Great Lakes Bioenergy Research Center, Michigan State University (MSU), ²Great Lakes Bioenergy Research Center and DOE-Plant Research Lab, Michigan State University (MSU) Plant biomass is a major carbon-neutral renewable resource that could be used for the production of biofuels. Plant biomass consists mainly of cell walls, a structurally complex composite material termed lignocellulosics. Here we describe a protocol for a comprehensive analysis of the content and composition of the polyphenolic lignin.

• Biology

  
  Primary Culture of Hippocampal Neurons from P0 Newborn Rats Joseph Nunez^{1 1}Department of Psychology, Michigan State University (MSU) The dissection and growth of cells from an individual brain area facilitates investigation of cellular and physiological parameters. We describe a method for primary cell culturing that produces neuron-enriched cultures in a serum-free environment.

• Biology

  
  Morris Water Maze Experiment Joseph Nunez^{1 1}Department of Psychology, Michigan State University (MSU) The Morris water maze is a well-accepted tool used to document the involvement of the hippocampus in a behavioral task.