University of Minnesota

73 articles published in JoVE

• Biology

  
  An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines Xin-Ru Wang¹, Nicole Y. Burkhardt¹, Lisa D. Price¹, Ulrike G. Munderloh^{1 1}Department of Entomology, University of Minnesota Electroporation is a rapid, broadly adopted method for introducing exogenous DNA into the genus Rickettsia. This protocol provides a useful electroporation method for the transformation of obligate intracellular bacteria in the genus Rickettsia.

• Engineering

  
  Microtensiometer for Confocal Microscopy Visualization of Dynamic Interfaces Steven V. Iasella¹, Sourav Barman¹, Clara Ciutara¹, Boxun Huang¹, Michael L. Davidson², Joseph A. Zasadzinski^{1 1}Department of Chemical Engineering and Materials Science, University of Minnesota, ²Department of Chemical Engineering, Carnegie Mellon University This manuscript describes the design and operation of a microtensiometer/confocal microscope to do simultaneous measurements of interfacial tension and surface dilatational rheology while visualizing the interfacial morphology. This provides the real-time construction of structure-property relationships of interfaces important in technology and physiology.

• Developmental Biology

  
  Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures Bridget T. Jacques-Fricke¹, Julaine Roffers-Agarwal^2,3, Callie M. Gustafson^2,3, Laura S. Gammill^{2,3 1}Department of Biology and Neuroscience Program, Hamline University, ²Department of Genetics, Cell Biology and Development, University of Minnesota, ³Developmental Biology Center, University of Minnesota This versatile protocol describes the isolation of premigratory neural crest cells (NCCs) through the excision of cranial neural folds from chick embryos. Upon plating and incubation, migratory NCCs emerge from neural fold explants, allowing for assessment of cell morphology and migration in a simplified 2D environment.

• Biology

  
  In Vivo Measurement of Hindlimb Dorsiflexor Isometric Torque from Pig Benjamin T. Corona¹, Jarrod A. Call^2,3, Matthew Borkowski⁴, Sarah M. Greising^{5 1}School of Medicine, Wake Forest University, ²Department of Kinesiology, University of Georgia, ³Regenerative Bioscience Center, University of Georgia, ⁴Aurora Scientific Inc., ⁵School of Kinesiology, University of Minnesota The present protocol describes concise experimental details on the evaluation and interpretation of in vivo torque data obtained via electrical stimulation of the common peroneal nerve in anesthetized pigs.

• Bioengineering

  
  Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection Gabriella Shull*^1,2, Christiane Haffner*³, Wieland B. Huttner³, Elena Taverna^3,4, Suhasa B. Kodandaramaiah^{1,5,6 1}Department of Biomedical Engineering, University of Minnesota, ²Department of Biomedical Engineering, Duke University, ³Max Planck Institute of Molecular Cell Biology and Genetics, ⁴Max Planck Institute for Evolutionary Anthropology, ⁵Department of Mechanical Engineering, University of Minnesota, ⁶Graduate Program in Neuroscience, University of Minnesota This protocol demonstrates the use of a robotic platform for microinjection into single neural stem cells and neurons in brain slices. This technique is versatile and offers a method of tracking cells in tissue with high spatial resolution.

• Neuroscience

  
  Cerebellar Regional Dissection for Molecular Analysis Katherine A. Hamel¹, Marija Cvetanovic^{1 1}Department of Neuroscience, University of Minnesota Different cerebellar regions have been implicated to play a role in distinct behavioral outputs, yet the underlying molecular mechanisms remain unknown. This work describes a method to reproducibly and quickly dissect cerebellar cortex of the hemispheres, anterior and posterior regions of the vermis, and the deep cerebellar nuclei in order to probe for molecular differences by isolating RNA and testing for differences in gene expression. 

• Biology

  
  Genome Engineering of Primary Human B Cells Using CRISPR/Cas9 Kanut Laoharawee^1,2,3, Matthew J. Johnson^1,2,3, Walker S. Lahr^1,2,3, Joseph J. Peterson^1,2,3, Beau R. Webber^1,2,3, Branden S. Moriarity^{1,2,3 1}Department of Pediatrics, University of Minnesota, ²Center for Genomic Engineering, University of Minnesota, ³Masonic Cancer Center, University of Minnesota Here we provide a detailed, step-by-step protocol for CRISPR/Cas9-based genome engineering of primary human B cells for gene knockout (KO) and knock-in (KI) to study biological functions of genes in B cells and the development of B-cell therapeutics.

• Biology

  
  Isolation of Cardiomyocytes from Fixed Hearts for Immunocytochemistry and Ploidy Analysis Doğacan Yücel*^1,2, Jacob Solinsky*², Jop H. van Berlo^{1,2,3 1}Department of Integrative Biology and Physiology, University of Minnesota, ²Lillehei Heart Institute, Department of Medicine, University of Minnesota, ³Stem Cell Institute, University of Minnesota The goal of this work is to develop a method to reproducibly isolate cardiomyocytes from the adult heart and measure DNA content and nucleation.

• Environment

  
  Microplot Design and Plant and Soil Sample Preparation for ¹⁵Nitrogen Analysis Jared A. Spackman¹, Fabian G. Fernandez^{1 1}Department of Soil, Water and Climate, University of Minnesota A microplot design for ¹⁵N tracer research is described to accommodate multiple in-season plant and soil sampling events. Soil and plant sample collection and processing procedures, including grinding and weighing protocols, for ¹⁵N analysis are put forth.

• Bioengineering

  
  Transduction and Expansion of Primary T Cells in Nine Days with Maintenance of Central Memory Phenotype Mary S. Pampusch¹, Pamela J. Skinner^{1 1}Department of Veterinary and Biomedical Sciences, University of Minnesota We outline a 9-day protocol for the transduction and expansion of rhesus macaque peripheral blood mononuclear cells which yields cells with excellent co-expression of the genes of interest in sufficient number for infusion studies of cell efficacy.

• Biochemistry

  
  In Vivo Calcium Imaging in C. elegans Body Wall Muscles Ashley A. Martin^1,2, Simon Alford³, Janet E. Richmond^{1 1}Department of Biological Sciences, University of Illinois at Chicago, ²Department of Integrative Biology and Physiology, University of Minnesota, ³Department of Anatomy and Cell Biology, University of Illinois at Chicago This method provides a way to couple optogenetics and genetically encoded calcium sensors to image baseline cytosolic calcium levels and changes in evoked calcium transients in the body wall muscles of the model organism C. elegans.

• Biology

  
  Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture Rebecca J. Morris¹, Nyssa Readio¹, Kelsey Boland¹, Kelly Johnson¹, Sonali Lad¹, Anupama Singh¹, Ashok Singh¹, Stephanie Holtorf¹, Samantha Skaar^{1 1}Hormel Institute, University of Minnesota The goal of this protocol is to isolate epidermal keratinocytes from the dorsal skin of adult mice for a variety of downstream applications such as molecular biology, biochemistry, fluorescence activated cell sorting, and primary in vitro uses (e.g., clonogenic keratinocytes).

• Biology

  
  SA-β-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs Heike Fuhrmann-Stroissnigg*¹, Fernando E. Santiago*^1,2,3, Diego Grassi¹, YuanYuan Ling¹, Laura J. Niedernhofer^1,2,3, Paul D. Robbins^{1,2,3 1}Department of Molecular Medicine and the Center on Aging, The Scripps Research Institute, ²Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, ³Institute on the Biology of Aging and Metabolism, University of Minnesota Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated β-Galactosidase activity in single cells.

• Medicine

  
  Spontaneous and Evoked Measures of Pain in Murine Models of Monoarticular Knee Pain Hollis E. Krug^1,2, Christopher Dorman², Nicole Blanshan², Sandra Frizelle², Maren Mahowald^{1,2 1}Department of Medicine, University of Minnesota, ²Research Department, VA Health Care Center We have developed an evoked measure of arthritis pain and coupled it with a standardized method for measuring spontaneous pain in different murine models of chemically induced arthritis. These measures are sensitive and reproducible for different types of joint pain.

• Engineering

  
  Fabrication of Three-Dimensional Graphene-Based Polyhedrons via Origami-Like Self-Folding Daeha Joung¹, Daniel Wratkowski¹, Chunhui Dai¹, Seokhyeong Lee¹, Jeong-Hyun Cho^{1 1}Department of Electrical and Computer Engineering, University of Minnesota, Minneapolis, United States Here, we present a protocol for fabrication of 3D graphene-based polyhedrons via origami-like self-folding.

• Environment

  
  Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn (Zea mays) As an Exemplar Carrie A. Deans^1,2, Gregory A. Sword¹, Paul A. Lenhart³, Eric Burkness², William D. Hutchison², Spencer T. Behmer^{1 1}Department of Entomology, Texas A&M University, ²Department of Entomology, University of Minnesota, ³Department of Entomology, University of Kentucky The protocols described herein provide a clear and approachable methodology for measuring soluble protein and digestible (non-structural) carbohydrate content in plant tissues. The ability to quantify these two plant macronutrients has significant implications for advancing the fields of plant physiology, nutritional ecology, plant-herbivore interactions and food-web ecology.

• Biochemistry

  
  Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues Dana M. Freund¹, Katherine A. Sammons², Nokwanda P. Makunga³, Jerry D. Cohen¹, Adrian D. Hegeman^{2 1}Department of Horticultural Science, Microbial and Plant Genomics Institute, University of Minnesota, ²Department of Horticultural Science, Department of Plant and Microbial Biology, Microbial and Plant Genomics Institute, University of Minnesota, ³Department of Botany and Zoology, Stellenbosch University Leaf spray mass spectrometry is a direct chemical analysis technique that minimizes the sample preparation and eliminates chromatography, allowing for the rapid detection of small molecules from plant tissues.

• Medicine

  
  Surgical Swine Model of Chronic Cardiac Ischemia Treated by Off-Pump Coronary Artery Bypass Graft Surgery Laura Hocum Stone¹, Christin Wright¹, Erin Chappuis¹, Mia Messer¹, Herbert B. Ward¹, Edward O. McFalls², Rosemary F. Kelly^{1 1}Department of Surgery, University of Minnesota, ²Cardiology, Minneapolis VA Medical Center This protocol presents a surgical large animal model of chronic, single vessel ischemia that results in regional abnormalities but does not create infarct, known as hibernating myocardium. Following establishment of chronic ischemia, animals are treated with off-pump LIMA-LAD coronary artery bypass graft surgery to revascularize the ischemic tissue.

• Genetics

  
  A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells Lisa N. Chesner¹, Colin Campbell^{1 1}Department of Pharmacology, University of Minnesota The goal of this protocol is to quantify the repair of defined DNA-protein crosslinks on plasmid DNA. Lesioned plasmids are transfected into recipient mammalian cell lines and low-molecular weight harvested at multiple time points post-transfection. DNA repair kinetics are quantified using strand-specific primer extension followed by qPCR.

• Medicine

  
  An Anatomical Study of Nerves at Risk During Minimally Invasive Hallux Valgus Surgery Miki Dalmau-Pastor^1,2,3, Jordi Vega^1,4, Francesc Malagelada^1,5, Fernando Peña⁶, Maria Cristina Manzanares-Céspedes^{1 1}Laboratory of Arthroscopic and Surgical Anatomy. Department of Pathology and Experimental Therapeutics (Human Anatomy and Embryology Unit), University of Barcelona, ²Health Sciences Faculty of Manresa, University of Vic-Central University of Catalunya, ³Groupe de Recherche et d'Etude en Chirurgie Mini-Invasive du Pied, GRECMIP, ⁴Foot and Ankle Unit, Hospital Quirón Barcelona, ⁵Foot and Ankle Unit, Orthopedic and Trauma Surgery, Royal London Hospital, Barts Health NHS Trust, ⁶Department of Orthopedic Surgery, Foot and Ankle Unit, University of Minnesota Minimally invasive surgical (MIS) procedures rely on anatomical references to localize structures not directly visible to the surgeon. This manuscript describes a combined method of plane-by-plane dissection and sectional anatomy of fresh-frozen specimens to locate the structures at risk during MIS procedures.

• Bioengineering

  
  Preparation, Purification, and Use of Fatty Acid-containing Liposomes Lin Jin*^1,2, Aaron E. Engelhart*^1,3, Katarzyna P. Adamala^1,3, Jack W. Szostak^{1 1}Howard Hughes Medical Institute and Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, ²Department of Biomedical Engineering, Boston University, ³Department of Genetics, Cell Biology, and Development, University of Minnesota Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

• Bioengineering

  
  Use of a Rat Model to Study Ventral Abdominal Hernia Repair Mark A. Suckow¹, Felicia D. Duke Boynton², Chad Johnson^{3 1}Department of Veterinary Population Medicine, University of Minnesota, ²Research Animal Resources, University of Minnesota, ³Cook Biotech, Inc. This manuscript describes methods associated with creation and repair of a ventral abdominal wall defect (hernia). This model can be used to study repair strategies such as those that use implanted materials. In this manuscript, repair of the experimental hernia with porcine small intestinal submucosa is presented as an example.

• Cancer Research

  
  A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s Erik S. Carlson^1,2, Pramod Upadhyaya², Stephen S. Hecht^{2 1}Department of Pharmacology, University of Minnesota, ²Masonic Cancer Center, University of Minnesota α-hydroxylation of carcinogenic nitrosamines by cytochrome P450s is the accepted metabolic pathway that produces DNA-damaging intermediates, which cause mutations. However, new data indicates further oxidation to nitrosamides can occur. We describe a general method for detecting nitrosamides produced from in vitro cytochrome P450-catalyzed metabolism of nitrosamines.

• Immunology and Infection

  
  In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues Shengbin Li¹, Gwantwa Mwakalundwa¹, Pamela J. Skinner^{1 1}Department of Veterinary and Biomedical Sciences, University of Minnesota Here, we describe a method that combines in situ MHC-tetramer staining with immunohistochemistry to determine localization, phenotype, and quantity of antigen-specific T cells in tissues. This protocol is used to determine the spatial and phenotypic characteristics of antigen-specific CD8 T cells relative to other cell type and structures in tissues.

• Neuroscience

  
  Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters Kelsey M. Moore¹, Brett T Himmler¹, Benjamin A Teplitzky², Matthew D Johnson^2,3, Robert L Meisel^{1 1}Department of Neuroscience, University of Minnesota, ²Department of Biomedical Engineering, University of Minnesota, ³Institute for Translational Neuroscience, University of Minnesota This paper details the use of fixed-potential amperometric recordings using carbon fiber electrodes and enzymatic biosensor technology to measure the release of dopamine and glutamate with high temporal resolution during natural rewarding behavior in the female hamster.

• Bioengineering

  
  Ammonia Synthesis at Low Pressure Edward Cussler¹, Alon McCormick¹, Michael Reese², Mahdi Malmali^{1 1}Department of Chemical Engineering and Materials Science, University of Minnesota – Twin Cities, ²West Central Research and Outreach Center, University of Minnesota – Morris Ammonia can be synthesized at low pressure by using a conventional catalyst and an ammonia selective absorbent.

• Bioengineering

  
  Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System Mark Rustad¹, Allen Eastlund², Ryan Marshall¹, Paul Jardine², Vincent Noireaux^{1 1}School of Physics and Astronomy, University of Minnesota, ²Department of Diagnostic and Biological Sciences and Institute for Molecular Virology, University of Minnesota A new generation of cell-free transcription-translation platforms has been engineered to construct biochemical systems in vitro through the execution of gene circuits. In this article, we describe how bacteriophages, such as MS2, ΦΧ174, and T7, are synthesized from their genome using an all E. coli cell-free TXTL system.

• Cancer Research

  
  Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis Charles E. Gast¹, Aubie K. Shaw^1,2, Melissa H. Wong^1,3, Lisa M. Coussens^{1,3 1}Cell, Developmental & Cancer Biology, Oregon Health & Science University, ²University of Minnesota, ³Knight Cancer Institute, Oregon Health & Science University Pre-clinical models evaluating adjuvant therapy targeting breast cancer metastasis are lacking. To address this, we developed a murine model of de novo pulmonary mammary adenocarcinoma metastasis, wherein therapies administered in the adjuvant setting (post surgical resection of primary tumors) can be evaluated for efficacy in impacting previously seeded pulmonary metastases.

• Genetics

  
  Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing Marisa E. Miller*^1,2, Katie L. Liberatore*^1,3, Shahryar F. Kianian^{1,3 1}Cereal Disease Laboratory, United States Department of Agriculture-Agricultural Research Service, ²Department of Horticultural Science, University of Minnesota, ³Department of Plant Pathology, University of Minnesota The comparison and optimization of two plant organellar DNA enrichment methods are presented: traditional differential centrifugation and fractionation of the total gDNA based on methylation status. We assess the resulting DNA quantity and quality, demonstrate performance in short-read next-generation sequencing, and discuss the potential for use in long-read single-molecule sequencing.

• Environment

  
  A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples Lawrence G. Oates¹, Harry W. Read², Jessica L. M. Gutknecht³, David S. Duncan¹, Teri B. Balser⁴, Randall D. Jackson^{1 1}Department of Agronomy and Great Lakes Bioenergy Research Center, University of Wisconsin - Madison, ²Department of Soil Science, University of Wisconsin - Madison, ³Department of Soil, Water, and Climate, University of Minnesota, ⁴Faculty of Science and Engineering, Curtin University The article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples.

• Developmental Biology

  
  Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles Abhijit Nirwane¹, Jyoti Gautam¹, Yao Yao^{1 1}College of Pharmacy, University of Minnesota This work describes a FACS-based protocol that allows for easy and simultaneous isolation of type I and type II pericytes from skeletal muscles.

• Neuroscience

  
  Fiber Connections of the Supplementary Motor Area Revisited: Methodology of Fiber Dissection, DTI, and Three Dimensional Documentation Baran Bozkurt¹, Kaan Yagmurlu², Erik H. Middlebrooks³, Zuzan Cayci⁴, Orhun M. Cevik¹, Ali Karadag⁵, Sean Moen¹, Necmettin Tanriover⁶, Andrew W. Grande^{1 1}Department of Neurosurgery, University of Minnesota, ²Department of Neurosurgery, Barrow Neurological Institute, St. Josephs Hospital and Medical Center, ³Department of Radiology, University of Alabama at Birmingham, ⁴Department of Radiology, University of Minnesota, ⁵Department of Neurosurgery, Tepecik Training and Research Hospital, ⁶Department of Neurosurgery, Cerrahpasa Medical School, University of Istanbul The purpose of this study is to show each step of the fiber dissection technique on human cadaveric brains, the 3D documentation of these dissections, and the diffusion tensor imaging of the anatomically dissected fiber pathways.

• Developmental Biology

  
  Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish Elaine L. Welch*¹, Celeste C. Eno*¹, Sreelaja Nair², Robin E. Lindeman³, Francisco Pelegri^{1 1}Laboratory of Genetics, University of Wisconsin-Madison, ²Department of Biological Sciences, Tata Institute of Fundamental Research, ³Department of Genetics, Cell Biology, and Development, University of Minnesota An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

• Cancer Research

  
  A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies Katherine A. Murphy*^1,2, Britnie R. James*^1,2,3, Andrew Wilber^4,5, Thomas S. Griffith^{1,2,3 1}Department of Urology, University of Minnesota, ²Masonic Cancer Center, University of Minnesota, ³Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota, ⁴Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, ⁵Simmons Cancer Institute Implementation of an orthotopic model of renal cell carcinoma in immunocompetent mice affords the investigator a clinically-relevant system defined by the presence of a primary renal tumor and lung metastases in the same animal. This system can be used to preclinically test a variety of treatments in vivo.

• Engineering

  
  Fabrication of Nanopillar-Based Split Ring Resonators for Displacement Current Mediated Resonances in Terahertz Metamaterials Chao Liu¹, Joseph Schauff¹, Seokhyeong Lee¹, Jeong-Hyun Cho^{1 1}Department of Electrical and Computer Engineering, University of Minnesota A protocol for the design and fabrication of a novel nanopillar-based split ring resonator (SRR) is presented.

• Genetics

  
  Generation of Fluorescent Protein Fusions in Candida Species Sara Gonia¹, Judith Berman², Cheryl A. Gale^{1 1}Department of Pediatrics, University of Minnesota, ²Department of Molecular Microbiology and Biotechnology, Tel Aviv University PCR-mediated gene modification can be used to generate fluorescent protein fusions in Candida species, which facilitates visualization and quantitation of yeast cells and proteins. Herein, we present a strategy for constructing a fluorescent protein fusion (Eno1-FP) in Candida parapsilosis.

• Biochemistry

  
  Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution Gatikrushna Singh*¹, Sarah M. Fritz*², Arnaz Ranji², Deepali Singh³, Kathleen Boris-Lawrie^{1,2 1}Department of Veterinary & Biomedical Sciences, University of Minnesota, ²Department of Veterinary Biosciences, Ohio State University, ³School of Biotechnology, Gautam Buddha University This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A.

• Bioengineering

  
  Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies Mary G. Garry¹, Stefan M. Kren¹, Daniel J. Garry^{1 1}Lillehei Heart Institute, University of Minnesota In these studies, we provide methodology for novel, neonatal, murine cardiac scaffolds for use in regenerative studies.

• Neuroscience

  
  Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity Cayla M. Duffy^1,2, Shihab Ahmed¹, Ce Yuan^1,2,3, Vijayakumar Mavanji¹, Joshua P. Nixon^1,2, Tammy Butterick^{1,2,4 1}Minneapolis Veterans Affairs Health Care System, ²Department of Food Science and Nutrition, University of Minnesota, ³Biomedical Informatics and Computational Biology Program, University of Minnesota, ⁴Minnesota Obesity Center, University of Minnesota Microglia (immune cells of the brain), are used as a surrogate biosensor to determine how nanoparticles influence neurotoxicity. We describe a series of experiments designed to assay microglial response to nanoparticles and exposure of hypothalamic neurons to supernatant from activated microglia to determine neurotoxicity.

• Bioengineering

  
  Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids Bradley P. Weegman¹, Ahmad Essawy², Peter Nash², Alexandra L. Carlson², Kristin J. Voltzke³, Zhaohui Geng², Marjan Jahani², Benjamin B. Becker², Klearchos K. Papas⁴, Meri T. Firpo^{2 1}Radiology, University of Minnesota, ²Medicine, University of Minnesota, ³School of Public Health, University of Minnesota, ⁴Surgery, University of Arizona Nutrient regulation using continuous growth adjusted feeding improves growth rates of mammalian cell spheroids compared to intermittent batch feeding for cultures in stirred suspension bioreactors. This study demonstrates the methods required for establishing simple adjusted rate fed cultures.

• Neuroscience

  
  Assays to Detect UV-reflecting Structures and Determine their Importance in Mate Preference using the Sailfin Molly Poecilia latipinna Shala J. Hankison¹, Meredith S. Palmer^{2 1}Department of Zoology, Ohio Wesleyan University, ²Department of Ecology, Evolution, & Behavior, University of Minnesota This protocol outlines the use of spectrophotometry to detect ultraviolet-reflecting structures on organisms (in this example, the sailfin molly Poecilia latipinna) and describes dichotomous choice tests for fish that allow inferences to be made on the role of ultraviolet cues during mate selection.

• Biology

  
  G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay Ansley Semack¹, Rabia U. Malik², Sivaraj Sivaramakrishnan^{1 1}Genetics, Cell Biology, and Development, University of Minnesota, ²Department of Cell and Developmental Biology, University of Michigan Simple methods to detect the selective activation of G proteins by G protein-coupled receptors remain an outstanding challenge in cell signaling. Here, Fӧrster resonance energy transfer (FRET) biosensors have been developed by pairwise tethering a GPCR to G protein peptides to probe conformational changes at controlled concentrations in live cells.

• Biology

  
  Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System Emanuel G. Worst¹, Matthias P. Exner², Alessandro De Simone², Marc Schenkelberger¹, Vincent Noireaux³, Nediljko Budisa², Albrecht Ott^{1 1}Department of Experimental Physics, Saarland University, ²Institute of Chemistry, Technische Universität Berlin, ³School of Physics and Astronomy, University of Minnesota An easy-to-use, cell-free expression protocol for the residue-specific incorporation of noncanonical amino acid analogs into proteins, including downstream analysis, is presented for medical, pharmaceutic, structural and functional studies.

• Medicine

  
  Testing the Efficacy of Pharmacological Agents in a Pericardial Target Delivery Model in the Swine Tinen L. Iles¹, Brian Howard², Stephen Howard³, Stephen Quallich², Christopher Rolfes², Eric Richardson⁴, Hanna R. Iaizzo⁵, Paul A. Iaizzo^{1 1}Surgery, University of Minnesota, ²Biomedical Engineering, University of Minnesota, ³Medtronic, Inc., ⁴Bioengineering, Rice University, ⁵Pharmacy, University of Wisconsin We have developed a swine model for the target delivery of pharmacological agents within the pericardial space/fluid. Using this approach, the relative benefits of administered agents on induced atrial fibrillation, relative refractory periods and/or ischemic protection can be investigated.

• Biology

  
  Measuring Pressure Volume Loops in the Mouse DeWayne Townsend^{1 1}Department of Integrative Biology and Physiology, University of Minnesota This manuscript describes a detailed protocol for the collection of pressure-volume data from the mouse.

• Immunology and Infection

  
  Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation Charles J. Benck¹, Tijana Martinov², Brian T. Fife², Devavani Chatterjea^{1 1}Department of Biology, Macalester College, ²Department of Medicine, Division of Rheumatic and Autoimmune Diseases, Center for Immunology, University of Minnesota This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

• Medicine

  
  A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting Eric B. Berens¹, Jon M. Holy², Anna T. Riegel¹, Anton Wellstein^{1 1}Lombardi Comprehensive Cancer Center, Department of Oncology, Georgetown University, ²Department of Biomedical Sciences, University of Minnesota This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

• Environment

  
  Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies Petra Kranzfelder¹, Alyssa M. Anderson², Alexander T. Egan¹, Jane E. Mazack¹, R. William Bouchard, Jr.³, Moriya M. Rufer⁴, Leonard C. Ferrington, Jr.^{1 1}Department of Entomology, University of Minnesota, ²Biology, Chemistry & Physics, and Mathematics Department, Northern State University, ³Environmental Analysis and Outcomes Division, Minnesota Pollution Control Agency, ⁴RMB Environmental Laboratories, Inc. Rapid bioassessment protocols using benthic macroinvertebrates are often used to monitor and assess water quality. An efficient protocol involves collections of Chironomidae surface-floating pupal exuviae (SFPE). Here, techniques for field collection, laboratory processing, slide mounting, and identification of Chironomidae SFPE are described.

• Bioengineering

  
  Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films Eric S. Hald¹, Kerianne E. Steucke¹, Jack A. Reeves¹, Zaw Win¹, Patrick W. Alford^{1 1}Department of Biomedical Engineering, University of Minnesota We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

• Behavior

  
  Morris Water Maze Test: Optimization for Mouse Strain and Testing Environment Daniel S. Weitzner¹, Elizabeth B. Engler-Chiurazzi², Linda A. Kotilinek³, Karen Hsiao Ashe^3,4,5, Miranda Nicole Reed^{1,6 1}Department of Psychology, Behavioral Neuroscience, West Virginia University, ²Department of Physiology and Pharmacology, West Virginia University, ³Department of Neurology, N. Bud Grossman Center for Memory Research and Care, University of Minnesota, ⁴Department of Neuroscience, N. Bud Grossman Center for Memory Research and Care, University of Minnesota, ⁵GRECC, VA Medical Center, ⁶Center for Neuroscience, Center for Basic and Translational Stroke Research, West Virginia University This manuscript describes a Morris water maze (MWM) protocol tailored for use with a commonly used mouse model of Alzheimer's disease. The MWM is widely used in transgenic mouse models. Implementation of a procedure sensitive to the background strain of the mouse model is essential for detecting group differences.

• Biology

  
  Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples Percy Griffin^1,2, Ashley Sexton³, Lauren Macneill³, Yoshie Iizuka^3,4, Michael K. Lee^1,2, Martina Bazzaro^{3,4 1}Department of Neuroscience, University of Minnesota, ²Institute for Translational Neuroscience, University of Minnesota, ³Department of Obstetrics, Gynecology, and Women’s Heath, University of Minnesota, ⁴Masonic Cancer Center, University of Minnesota The current protocol details a method for measuring the activity of functionally homologous deubiquitinating enzymes. Specialized probes covalently modify the enzyme and allow for detection. This method holds the potential to identify new therapeutic targets.

• Immunology and Infection

  
  High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization Jana Ninković^1,3, Sabita Roy^{1,2 1}Department of Pharmacology, University of Minnesota, ²Department of Surgery, University of Minnesota, ³3M Corporate Research Laboratory Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

• Neuroscience

  
  The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism Sara Tremblay¹, Vincent Beaulé¹, Sébastien Proulx², Louis-Philippe Lafleur¹, Julien Doyon¹, Małgorzata Marjańska³, Hugo Théoret^{1 1}Department of Psychology, University of Montréal, ²Montreal Neurological Institute, McGill University, ³Center for Magnetic Resonance Research and Department of Radiology, University of Minnesota This article aims to describe a basic protocol for combining transcranial direct current stimulation (tDCS) with proton magnetic resonance spectroscopy (¹H-MRS) measurements to investigate the effects of bilateral stimulation on primary motor cortex metabolism.

• Bioengineering

  
  CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells Jing Ge*¹, Somsak Prasongtanakij*², David K. Wood³, David M. Weingeist¹, Jessica Fessler¹, Panida Navasummrit², Mathuros Ruchirawat², Bevin P. Engelward^{1 1}Department of Biological Engineering, Massachusetts Institute of Technology, ²Environmental Toxicology, Chulabhorn Graduate Institute, ³Department of Biomedical Engineering, University of Minnesota We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics.

• Immunology and Infection

  
  Isolation, Identification, and Purification of Murine Thymic Epithelial Cells Yan Xing¹, Kristin A. Hogquist^{1 1}Department of Laboratory Medicine & Pathology, Center for Immunology, University of Minnesota Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

• Medicine

  
  Shrinkage of Dental Composite in Simulated Cavity Measured with Digital Image Correlation Jianying Li¹, Preetanjali Thakur¹, Alex S. L. Fok^{1 1}Minnesota Dental Research Center for Biomaterials and Biomechanics, School of Dentistry, University of Minnesota In order to understand the spatial development of polymerization shrinkage stress in dental resin-composite restorations, Digital Image Correlation was used to provide full-field displacement/strain measurement of restored model glass cavities by correlating images of the restoration taken before and after polymerization.

• Bioengineering

  
  Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method Nathan J. Wittenberg¹, Timothy W. Johnson¹, Luke R. Jordan², Xiaohua Xu³, Arthur E. Warrington³, Moses Rodriguez^3,4, Sang-Hyun Oh^{1,2 1}Department of Electrical and Computer Engineering, University of Minnesota, ²Department of Biomedical Engineering, University of Minnesota, ³Department of Neurology, Mayo Clinic College of Medicine, ⁴Department of Immunology, Mayo Clinic College of Medicine Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

• Neuroscience

  
  Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model Tammy A. Butterick^1,2, Cayla M. Duffy^1,2, Rachel E. Lee³, Charles J. Billington^1,2,4, Catherine M. Kotz^1,2, Joshua P. Nixon^{1,2 1}Department of Veterans Affairs, Minneapolis Veterans Affairs Health Care System, ²Department of Food Science and Nutrition, University of Minnesota, ³Department of Integrative Biology and Physiology, University of Minnesota, ⁴Department of Medicine, University of Minnesota Medical School, University of Minnesota Multiplex assays can provide beneficial information for basic cellular mechanisms and eliminate waste of reagents and unnecessary repetitive experiments. We describe here a multiplex caspase-3/7 activity assay, using fluorescent- and luminescent-based methods, to determine cell viability in an in vitro hypothalamic model following oxidative challenge with palmitic acid.

• Biology

  
  Mouse Genome Engineering Using Designer Nucleases Mario Hermann¹, Tomas Cermak², Daniel F. Voytas², Pawel Pelczar^{1 1}Institute of Laboratory Animal Science, University of Zurich, ²Department of Genetics, Cell Biology & Development and Center for Genome Engineering, University of Minnesota Designer nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to modify the genome of mouse preimplantation embryos by triggering both the nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways. These advances enable the rapid generation of mice with precise genetic modifications.

• Medicine

  
  Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens Lee J. Pribyl¹, Kathleen A. Coughlin¹, Thanasak Sueblinvong¹, Kristin Shields², Yoshie Iizuka^1,3, Levi S. Downs^1,3, Rahel G. Ghebre^1,3, Martina Bazzaro^{1,3 1}Department of Obstetrics, Gynecology, and Women's Heath, University of Minnesota, ²Department of Obstetrics and Gynecology, Maricopa Medical Center and St Josephs Hospital and Medical Center, ³Masonic Cancer Center, University of Minnesota This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

• Biology

  
  Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology Zachary Z. Sun*¹, Clarmyra A. Hayes*², Jonghyeon Shin³, Filippo Caschera⁴, Richard M. Murray², Vincent Noireaux^{4 1}Department of Biology, California Institute of Technology, ²Department of Bioengineering, California Institute of Technology, ³Synthetic Biology Center, Department of Bioengineering, Massachusetts Institute of Technology, ⁴School of Physics and Astronomy, University of Minnesota This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

• Bioengineering

  
  Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs) Allison M. Bock*^1,2, David Knorr*^1,2, Dan S. Kaufman^{1,2 1}Department of Medicine (Hematology, Oncology, and Transplant), University of Minnesota, Minneapolis, ²Stem Cell Institute, University of Minnesota, Minneapolis This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

• Bioengineering

  
  Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens Julianne Spencer^1,2, Emily Fitch³, Paul A. Iaizzo^{1,2,4,5 1}Department of Surgery, University of Minnesota, ²Department of Biomedical Engineering, University of Minnesota, ³Department of Biology, University of Minnesota, ⁴Department of Integrative Biology & Physiology, University of Minnesota, ⁵Institute for Engineering in Medicine, University of Minnesota The objective of this research is to recreate and then access the anatomy of the human cardiac venous system using 3D reconstructions generated from contrast-computed tomography scans.

• Medicine

  
  Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR Callie L. Janik^1,2, Timothy K. Starr^{1,2 1}Department of Obstetrics, Gynecology & Women's Health, Masonic Cancer Center, University of Minnesota, Minneapolis, ²Department of Genetics, Cell Biology & Development, Center for Genome Engineering, University of Minnesota, Minneapolis A method of identifying unknown drivers of carcinogenesis using an unbiased approach is described. The method uses the Sleeping Beauty transposon as a random mutagen directed to specific tissues. Genomic mapping of transposon insertions that drive tumor formation identifies novel oncogenes and tumor suppressor genes

• Neuroscience

  
  Creating Objects and Object Categories for Studying Perception and Perceptual Learning Karin Hauffen^1,2,3, Eugene Bart⁴, Mark Brady⁵, Daniel Kersten⁶, Jay Hegdé^{1,2,3 1}Brain and Behavior Discovery Institute, Georgia Health Sciences University, ²Vision Discovery Institute, Georgia Health Sciences University, ³Department of Opthalmology, Georgia Health Sciences University, ⁴Intelligent Systems Laboratory, Palo Alto Research Center, ⁵Pattern Recognition Systems, Palo Alto Research Center, ⁶Department of Psychology, University of Minnesota We describe a novel methodology for creating naturalistic 3-D objects and object categories with precisely defined feature variations. We use simulations of the biological processes of morphogenesis and phylogenesis to create novel, naturalistic virtual 3-D objects and object categories that can then be rendered as visual images or haptic objects.

• Biology

  
  Performing Custom MicroRNA Microarray Experiments Xiaoxiao Zhang¹, Yan Zeng^{1,2 1}Department of Pharmacology, University of Minnesota, ²Masonic Cancer Center, University of Minnesota A simple procedure of performing custom microRNA microarray experiments is described. The steps include isolating RNA, labeling RNA and reference DNA, hybridizing the samples to microarrays, scanning the microarrays, quantifying and analyzing hybridization signals.

• Biology

  
  Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica John D. Chan¹, Jonathan S. Marchant^{1 1}Department of Pharmacology and The Stem Cell Institute, University of Minnesota Medical School An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.

• Immunology and Infection

  
  Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field John Y. Takekawa¹, Nichola J. Hill^1,2, Annie K. Schultz¹, Samuel A. Iverson¹, Carol J. Cardona^3,4, Walter M. Boyce², Joseph P. Dudley^{5 1}USGS Western Ecological Research Center, ²Wildlife Health Center, University of California, Davis, ³Department of Population Health and Reproduction, University of California, Davis, ⁴Department of Veterinary and Biomedical Sciences, University of Minnesota, ⁵Science Applications International Corporation This study describes diagnosis of avian influenza in wild birds using a portable rRT-PCR system. The method takes advantage of freeze-dried reagents to screen wild birds in a non-laboratory setting, typical of an outbreak scenario. Use of molecular tools provides accurate and sensitive alternatives for rapid diagnosis.

• Neuroscience

  
  Labeling F-actin Barbed Ends with Rhodamine-actin in Permeabilized Neuronal Growth Cones Bonnie M. Marsick¹, Paul C. Letourneau^{1 1}Department of Neuroscience, University of Minnesota A method to visualize and quantify F-actin barbed ends in neuronal growth cones is described. After culturing neurons on glass coverslips, cells are permeabilized with a saponin-containing solution. Then, a short incubation with the saponin buffer containing rhodamine-actin incorporates fluorescent actin onto free actin barbed ends.

• Biology

  
  Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi Jonathan S. Schilling¹, K. Brook Jacobson*^{1 1}Department of Bioproducts and Biosystems Engineering, University of Minnesota This video demonstrates a controlled environment approach to study degradation of lignocellulosic plant tissues by aerobic fungi. The ability to control nutrient sources and moisture is a key advantage of agar-block microcosms, but the approach often yields mixed success. We address critical pitfalls to yield reproducible, low-variability results.

• Neuroscience

  
  An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells Tiffany M Schmidt¹, Paulo Kofuji^{1 1}Department of Neuroscience, University of Minnesota This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

• Biology

  
  A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ Qingsheng Li¹, Pamela J. Skinner², Lijie Duan¹, Ashley T. Haase^{1 1}Department of Microbiology, Medical School, University of Minnesota, ²Department of Veterinary and Biomedical Sciences, University of Minnesota A technique combining in situ tetramer staining and in situ hybridization (ISTH) enables visualization, mapping and analysis of the spatial proximity of virus-specific CD8+ T cells to their virus-infected targets, and determination of the quantitative relationships between these immune effectors and targets to infection outcomes.

• Biology

  
  Gibberella zeae Ascospore Production and Collection for Microarray Experiments. Matias Pasquali^1,2, Corby Kistler^{3 1}Cereal Disease Laboratory, USDA, ²University of Minnesota/ Agroinnova, University of Torino, ³Cereal Disease Laboratory, University of Minnesota To study the developmental processes of ascospores in Gibberella zeae, a procedure for collection under sterile conditions is filmed in order to generate the highest level of information for protocol description. This should facilitate the reproducibility of the experiment, a crucial aspect when full genome expression profile tests are implemented.