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88 ARTICLES PUBLISHED IN JoVE

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Biology

Gibberella zeae Ascospore Production and Collection for Microarray Experiments.
Matias Pasquali 1,2, Corby Kistler 3
1Cereal 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.

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Biology

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ
Qingsheng Li 1, Pamela J. Skinner 2, Lijie Duan 1, Ashley T. Haase 1
1Department 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.

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Biology

Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi
Jonathan S. Schilling 1, K. Brook Jacobson *1
1Department 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.

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Neuroscience

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells
Tiffany M Schmidt 1, Paulo Kofuji 1
1Department 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.

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Neuroscience

Labeling F-actin Barbed Ends with Rhodamine-actin in Permeabilized Neuronal Growth Cones
Bonnie M. Marsick 1, Paul C. Letourneau 1
1Department 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.

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Advanced Biology

ELISA Assays: Indirect, Sandwich, and Competitive
Whitney Swanson 1,2, Frances V. Sjaastad 2,3, Thomas S. Griffith 1,2,3,4
1Department of Urology, University of Minnesota, 2Center for Immunology, University of Minnesota, 3Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota, 4Masonic Cancer Center, University of Minnesota

ELISA Assays: Indirect, Sandwich, and Competitive

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Advanced Biology

Antibody Generation: Producing Monoclonal Antibodies Using Hybridomas
Frances V. Sjaastad 1,2, Whitney Swanson 2,3, Thomas S. Griffith 1,2,3,4
1Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota, 2Center for Immunology, University of Minnesota, 3Department of Urology, University of Minnesota, 4Masonic Cancer Center, University of Minnesota

Antibody Generation: Producing Monoclonal Antibodies Using Hybridomas

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Advanced Biology

Immunofluorescence Microscopy: Immunofluorescence Staining of Paraffin-Embedded Tissue Sections
Thomas Chaffee 1, Thomas S. Griffith 2,3,4, Kathryn L. Schwertfeger 1,3,4
1Department of Lab Medicine and Pathology, University of Minnesota, 2Department of Urology, University of Minnesota, 3Masonic Cancer Center, University of Minnesota, 4Center for Immunology, University of Minnesota

Immunofluorescence Microscopy: Immunofluorescence Staining of Paraffin-Embedded Tissue Sections

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Advanced Biology

Assay for Cell Death: Chromium Release Assay of Cytotoxic Ability
Frances V. Sjaastad 1,2, Whitney Swanson 2,3, Thomas S. Griffith 1,2,3,4
1Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota, 2Center for Immunology, University of Minnesota, 3Department of Urology, University of Minnesota, 4Masonic Cancer Center, University of Minnesota

Assay for Cell Death: Chromium Release Assay of Cytotoxic Ability

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Bioengineering

Anatomical Reconstructions of the Human Cardiac Venous System using Contrast-computed Tomography of Perfusion-fixed Specimens
Julianne Spencer 1,2, Emily Fitch 3, Paul A. Iaizzo 1,2,4,5
1Department 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.

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Bioengineering

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells
Jing Ge *1, Somsak Prasongtanakij *2, David K. Wood 3, David M. Weingeist 1, Jessica Fessler 1, Panida Navasummrit 2, Mathuros Ruchirawat 2, Bevin P. Engelward 1
1Department 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.

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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 Shin 3, Filippo Caschera 4, Richard M. Murray 2, Vincent Noireaux 4
1Department 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.

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Biology

Mouse Genome Engineering Using Designer Nucleases
Mario Hermann 1, Tomas Cermak 2, Daniel F. Voytas 2, Pawel Pelczar 1
1Institute 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.

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Medicine

Shrinkage of Dental Composite in Simulated Cavity Measured with Digital Image Correlation
Jianying Li 1, Preetanjali Thakur 1, Alex S. L. Fok 1
1Minnesota 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.

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Neuroscience

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus
Tijana Martinov 1, Madison Mack 1, Akilah Sykes 1, Devavani Chatterjea 1
1Biology Department, Macalester College

Electronic von Frey measurements are an effective and improved alternative to classical von Frey filaments, as they allow rapid and precise measurement of changes in rodent mechanical withdrawal thresholds to continuously applied pressure stimuli before and after an inflammatory event.

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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. Lee 3, Charles J. Billington 1,2,4, Catherine M. Kotz 1,2, Joshua P. Nixon 1,2
1Department 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.

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Bioengineering

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method
Nathan J. Wittenberg 1, Timothy W. Johnson 1, Luke R. Jordan 2, Xiaohua Xu 3, Arthur E. Warrington 3, Moses Rodriguez 3,4, Sang-Hyun Oh 1,2
1Department 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.

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Medicine

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens
Lee J. Pribyl 1, Kathleen A. Coughlin 1, Thanasak Sueblinvong 1, Kristin Shields 2, Yoshie Iizuka 1,3, Levi S. Downs 1,3, Rahel G. Ghebre 1,3, Martina Bazzaro 1,3
1Department 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.

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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 Tremblay 1, Vincent Beaulé 1, Sébastien Proulx 2, Louis-Philippe Lafleur 1, Julien Doyon 1, Małgorzata Marjańska 3, Hugo Théoret 1
1Department 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.

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Immunology and Infection

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers
Chandirasegaran Massilamany 1, Arunakumar Gangaplara 1, Ting Jia 1, Christian Elowsky 2, Qingsheng Li 3, You Zhou 2, Jay Reddy 1
1School of Veterinary Medicine and Biomedical Sciences, University of Nebraska, Lincoln, 2Center for Biotechnology, University of Nebraska, Lincoln, 3Nebraska Center for Virology and School of Biological Sciences, University of Nebraska, Lincoln

The protocol to detect self-reactive CD4 T cells in brain and heart by direct staining with major histocompatibility complex class II dextramers has been described in this report. For comprehensive analysis, a reliable method to enumerate the frequencies of antigen-specific CD4+ T cells in situ is also devised.

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Immunology and Infection

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells
Yan Xing 1, Kristin A. Hogquist 1
1Department 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.

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Immunology and Infection

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization
Jana Ninković 1,3, Sabita Roy 1,2
1Department 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.

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Bioengineering

Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids
Bradley P. Weegman 1, Ahmad Essawy 2, Peter Nash 2, Alexandra L. Carlson 2, Kristin J. Voltzke 3, Zhaohui Geng 2, Marjan Jahani 2, Benjamin B. Becker 2, Klearchos K. Papas 4, Meri T. Firpo 2
1Radiology, 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.

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Environment

Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies
Petra Kranzfelder 1, Alyssa M. Anderson 2, Alexander T. Egan 1, Jane E. Mazack 1, R. William Bouchard, Jr. 3, Moriya M. Rufer 4, Leonard C. Ferrington, Jr. 1
1Department 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.

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JoVE Core

Testing the Efficacy of Pharmacological Agents in a Pericardial Target Delivery Model in the Swine
Tinen L. Iles 1, Brian Howard 2, Stephen Howard 3, Stephen Quallich 2, Christopher Rolfes 2, Eric Richardson 4, Hanna R. Iaizzo 5, Paul A. Iaizzo 1
1Surgery, 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.

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Biology

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples
Percy Griffin 1,2, Ashley Sexton 3, Lauren Macneill 3, Yoshie Iizuka 3,4, Michael K. Lee 1,2, Martina Bazzaro 3,4
1Department 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.

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Bioengineering

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films
Eric S. Hald 1, Kerianne E. Steucke 1, Jack A. Reeves 1, Zaw Win 1, Patrick W. Alford 1
1Department 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.

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Medicine

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting
Eric B. Berens 1, Jon M. Holy 2, Anna T. Riegel 1, Anton Wellstein 1
1Lombardi 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.

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Bioengineering

Use of a Rat Model to Study Ventral Abdominal Hernia Repair
Mark A. Suckow 1, Felicia D. Duke Boynton 2, Chad Johnson 3
1Department 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.

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Immunology and Infection

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation
Charles J. Benck 1, Tijana Martinov 2, Brian T. Fife 2, Devavani Chatterjea 1
1Department 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.

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Biology

Measuring Pressure Volume Loops in the Mouse
DeWayne Townsend 1
1Department of Integrative Biology and Physiology, University of Minnesota

This manuscript describes a detailed protocol for the collection of pressure-volume data from the mouse.

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Biology

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System
Emanuel G. Worst 1, Matthias P. Exner 2, Alessandro De Simone 2, Marc Schenkelberger 1, Vincent Noireaux 3, Nediljko Budisa 2, Albrecht Ott 1
1Department 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.

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Biochemistry

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution
Gatikrushna Singh *1, Sarah M. Fritz *2, Arnaz Ranji 2, Deepali Singh 3, Kathleen Boris-Lawrie 1,2
1Department 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.

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Neuroscience

Assays to Detect UV-reflecting Structures and Determine their Importance in Mate Preference using the Sailfin Molly Poecilia latipinna
Shala J. Hankison 1, Meredith S. Palmer 2
1Department 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.

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Bioengineering

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies
Mary G. Garry 1, Stefan M. Kren 1, Daniel J. Garry 1
1Lillehei Heart Institute, University of Minnesota

In these studies, we provide methodology for novel, neonatal, murine cardiac scaffolds for use in regenerative studies.

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Neuroscience

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity
Cayla M. Duffy 1,2, Shihab Ahmed 1, Ce Yuan 1,2,3, Vijayakumar Mavanji 1, Joshua P. Nixon 1,2, Tammy Butterick 1,2,4
1Minneapolis 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.

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Biology

G Protein-selective GPCR Conformations Measured Using FRET Sensors in a Live Cell Suspension Fluorometer Assay
Ansley Semack 1, Rabia U. Malik 2, Sivaraj Sivaramakrishnan 1
1Genetics, 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.

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Cancer Research

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis
Charles E. Gast 1, Aubie K. Shaw 1,2, Melissa H. Wong 1,3, Lisa M. Coussens 1,3
1Cell, 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.

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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
1Department 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.

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Developmental Biology

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish
Elaine L. Welch *1, Celeste C. Eno *1, Sreelaja Nair 2, Robin E. Lindeman 3, Francisco Pelegri 1
1Laboratory 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.

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JoVE Core

Fabrication of Nanopillar-Based Split Ring Resonators for Displacement Current Mediated Resonances in Terahertz Metamaterials
Chao Liu 1, Joseph Schauff 1, Seokhyeong Lee 1, Jeong-Hyun Cho 1
1Department 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.

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Environment

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples
Lawrence G. Oates 1, Harry W. Read 2, Jessica L. M. Gutknecht 3, David S. Duncan 1, Teri B. Balser 4, Randall D. Jackson 1
1Department 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.

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Genetics

Generation of Fluorescent Protein Fusions in Candida Species
Sara Gonia 1, Judith Berman 2, Cheryl A. Gale 1
1Department 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.

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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
1Cereal 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.

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Neuroscience

Fiber Connections of the Supplementary Motor Area Revisited: Methodology of Fiber Dissection, DTI, and Three Dimensional Documentation
Baran Bozkurt 1, Kaan Yagmurlu 2, Erik H. Middlebrooks 3, Zuzan Cayci 4, Orhun M. Cevik 1, Ali Karadag 5, Sean Moen 1, Necmettin Tanriover 6, Andrew W. Grande 1
1Department 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.

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Biology

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci
Dan-Tong Zhu 1, Xin-Ru Wang 1, Fei-Xue Ban 1, Chi Zou 1, Shu-Sheng Liu 1, Xiao-Wei Wang 1
1Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogen and Insects, Institute of Insect Sciences, Zhejiang University

Here, we present a protocol to isolate endosymbionts from the whitefly Bemisia tabaci through dissection and filtration. After amplification, the DNA samples are suitable for subsequent sequencing and study of the mutualism between endosymbionts and whitefly.

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Developmental Biology

Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles
Abhijit Nirwane 1, Jyoti Gautam 1, Yao Yao 1
1College 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.

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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 Li 1, Gwantwa Mwakalundwa 1, Pamela J. Skinner 1
1Department 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.

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Neuroscience

Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters
Kelsey M. Moore 1, Brett T Himmler 1, Benjamin A Teplitzky 2, Matthew D Johnson 2,3, Robert L Meisel 1
1Department 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.

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Bioengineering

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System
Mark Rustad 1, Allen Eastlund 2, Ryan Marshall 1, Paul Jardine 2, Vincent Noireaux 1
1School 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.

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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 Peña 6, Maria Cristina Manzanares-Céspedes 1
1Laboratory 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.

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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 Upadhyaya 2, Stephen S. Hecht 2
1Department 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.

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Medicine

Surgical Swine Model of Chronic Cardiac Ischemia Treated by Off-Pump Coronary Artery Bypass Graft Surgery
Laura Hocum Stone 1, Christin Wright 1, Erin Chappuis 1, Mia Messer 1, Herbert B. Ward 1, Edward O. McFalls 2, Rosemary F. Kelly 1
1Department 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.

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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
1Howard 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.

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Genetics

A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells
Lisa N. Chesner 1, Colin Campbell 1
1Department 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.

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Biochemistry

Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues
Dana M. Freund 1, Katherine A. Sammons 2, Nokwanda P. Makunga 3, Jerry D. Cohen 1, Adrian D. Hegeman 2
1Department 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.

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Biology

SA-β-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs
Heike Fuhrmann-Stroissnigg *1, Fernando E. Santiago *1,2,3, Diego Grassi 1, YuanYuan Ling 1, Laura J. Niedernhofer 1,2,3, Paul D. Robbins 1,2,3
1Department 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.

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Environment

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn (Zea mays) As an Exemplar
Carrie A. Deans 1,2, Gregory A. Sword 1, Paul A. Lenhart 3, Eric Burkness 2, William D. Hutchison 2, Spencer T. Behmer 1
1Department 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.

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Engineering

Fabrication of Three-Dimensional Graphene-Based Polyhedrons via Origami-Like Self-Folding
Daeha Joung 1, Daniel Wratkowski 1, Chunhui Dai 1, Seokhyeong Lee 1, Jeong-Hyun Cho 1
1Department 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.

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Biology

Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture
Rebecca J. Morris 1, Nyssa Readio 1, Kelsey Boland 1, Kelly Johnson 1, Sonali Lad 1, Anupama Singh 1, Ashok Singh 1, Stephanie Holtorf 1, Samantha Skaar 1
1Hormel 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).

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Medicine

Spontaneous and Evoked Measures of Pain in Murine Models of Monoarticular Knee Pain
Hollis E. Krug 1,2, Christopher Dorman 2, Nicole Blanshan 2, Sandra Frizelle 2, Maren Mahowald 1,2
1Department 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.

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Biochemistry

In Vivo Calcium Imaging in C. elegans Body Wall Muscles
Ashley A. Martin 1,2, Simon Alford 3, Janet E. Richmond 1
1Department 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.

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Biochemistry

A Double Humanized BLT-mice Model Featuring a Stable Human-Like Gut Microbiome and Human Immune System
Lance Daharsh 1,2, Jianshui Zhang 1,2, Amanda Ramer-Tait 3, Qingsheng Li 1,2
1Nebraska Center for Virology, 2School of Biological Sciences, University of Nebraska-Lincoln, 3Department of Food Science and Technology, University of Nebraska-Lincoln

We describe a novel method for generating double humanized BLT-mice that feature a functional human immune system and a stable engrafted human-like gut microbiome. This protocol can be followed without the need for germ-free mice or gnotobiotic facilities.

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Bioengineering

Transduction and Expansion of Primary T Cells in Nine Days with Maintenance of Central Memory Phenotype
Mary S. Pampusch 1, Pamela J. Skinner 1
1Department 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.

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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
1Department 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.

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Environment

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis
Jared A. Spackman 1, Fabian G. Fernandez 1
1Department 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.

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Neuroscience

Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging
Hossein Nazari 1,2, Maxim Ivannikov 2, Lorenzo Ochoa 2,3, Gracie Vargas 2,3, Massoud Motamedi 3,4
1Department of Ophthalmology and Visual Neuroscience, University of Minnesota, 2Department of Neuroscience, Cell Biology, and Anatomy, University of Texas Medical Branch, 3Biomedical Engineering and Imaging Sciences Group, University of Texas Medical Branch, 4Department of Ophthalmology, University of Texas Medical Branch

Presented here is a protocol for intact whole retina imaging in which the outer opaque/pigmented layers of the eyeball are surgically removed, and optical clearing is applied to render retina transparent enabling the visualization of the peripheral retina and hyaloid vasculature in intact retina using light sheet fluorescent microscopy.

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Bioengineering

Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection
Gabriella Shull *1,2, Christiane Haffner *3, Wieland B. Huttner 3, Elena Taverna 3,4, Suhasa B. Kodandaramaiah 1,5,6
1Department 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.

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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
1Department 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.

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Neuroscience

Cerebellar Regional Dissection for Molecular Analysis
Katherine A. Hamel 1, Marija Cvetanovic 1
1Department 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. 

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Neuroscience

Detecting Amyloid-β Accumulation via Immunofluorescent Staining in a Mouse Model of Alzheimer's Disease
Zijian Song *1, Miao Zheng *1, Jiahui Ding 1, Yidi Xu 1, Miao-Jin Ji 1, Chao Liu 1
1Jiangsu Province Key Laboratory of Anesthesiology, Jiangsu Province Key Laboratory of Anesthesia and Analgesia Application Technology and NMPA Key Laboratory for Research and Evaluation of Narcotic and Psychotropic Drugs, Xuzhou Medical University

In the neuropathology of Alzheimer's disease, one of the most crucial characteristics is the deposition of amyloid-β. In this protocol, we describe the method of immunofluorescent staining in 5×FAD transgenic mouse to detect amyloid-β accumulation in plaques. The process of perfusion, cryosectioning, staining and quantification will be described in detail.

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Biology

In Vivo Measurement of Hindlimb Dorsiflexor Isometric Torque from Pig
Benjamin T. Corona 1, Jarrod A. Call 2,3, Matthew Borkowski 4, Sarah M. Greising 5
1School 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.

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Developmental Biology

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures
Bridget T. Jacques-Fricke 1, Julaine Roffers-Agarwal 2,3, Callie M. Gustafson 2,3, Laura S. Gammill 2,3
1Department 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.

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Engineering

Microtensiometer for Confocal Microscopy Visualization of Dynamic Interfaces
Steven V. Iasella 1, Sourav Barman 1, Clara Ciutara 1, Boxun Huang 1, Michael L. Davidson 2, Joseph A. Zasadzinski 1
1Department 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.

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Neuroscience

Imaging the Aging Cochlea with Light-Sheet Fluorescence Microscopy
Peter A. Santi 1, Shane B. Johnson 1
1Department of Otolaryngology, University of Minnesota

A light-sheet microscope was developed to image and digitize whole cochlea.

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Biology

Tick Artificial Membrane Feeding for Ixodes scapularis
Benedict Khoo 1, Benjamin Cull 2, Jonathan D. Oliver 1
1Division of Environmental Health Sciences, School of Public Health, University of Minnesota, 2Department of Entomology, College of Food, Agricultural and Natural Resources, University of Minnesota

Presented here is a method to blood feed ticks in vitro via an artificial membrane system to allow for partial or full engorgement of a variety of tick life stages.

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Biology

An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines
Xin-Ru Wang 1, Nicole Y. Burkhardt 1, Lisa D. Price 1, Ulrike G. Munderloh 1
1Department 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.

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Methods for the Study of Ticks, Mosquitoes, and their Transmitted Pathogens: Toward a Greater Understanding of Vector Biology and Arthropod-Microbe Interactions
Benjamin Cull *1, Xin-Ru Wang *1
1Department of Entomology, University of Minnesota

Methods for the Study of Ticks, Mosquitoes, and their Transmitted Pathogens: Toward a Greater Understanding of Vector Biology and Arthropod-Microbe Interactions

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Biology

Evidence for EpCAM and Cytokeratin Expressing Epithelial Cells in Normal Human and Murine Blood and Bone Marrow
Stephanie M. Holtorf 1, Jennifer Boyle 1, Rebecca Morris 1
1The Hormel Institute, University of Minnesota

This paper presents a reproducible method with new findings on the presence of epithelial cells in normal human and mouse blood and bone marrow using flow cytometry and immunofluorescence microscopy. Krt1-14;mTmG transgenic mice were used as an in vivo method to confirm these findings.

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Biology

Rearing the Cabbage White Butterfly (Pieris rapae) in Controlled Conditions: A Case Study with Heavy Metal Tolerance
Emilie C. Snell-Rood 1, Megan E. Kobiela 1,2
1Deptartment of Ecology, Evolution and Behavior, University of Minnesota, 2Biology, Sweet Briar College

This paper presents a detailed protocol for rearing the cabbage white butterfly in controlled lab conditions with an artificial diet, which allows precise manipulations of early-life nutrition and toxin exposure. The representative results show how heavy metal toxicity can be assayed with this protocol.

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Medicine

Technical Considerations and Approach to Redo Foregut Surgery
David J. Leishman 1, Divinemercy Bakare 2, Madhuri Rao 1
1Department of Surgery, Division of Thoracic and Foregut Surgery, University of Minnesota, 2University of Minnesota Medical School

Redo foregut surgery is associated with increased patient morbidity and presents a technical challenge for the surgeon. We describe our approach and considerations when performing a redo hiatal hernia repair to provide a guide for other surgeons and improve patient outcomes.

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Medicine

Surgical Porcine Model of Chronic Myocardial Ischemia Treated by Exosome-laden Collagen Patch and Off-pump Coronary Artery Bypass Graft
Rishav Aggarwal 1, Annie Shao 1, Koray N. Potel 2, Laura Hocum Stone 1, Cory Swingen 1, Christin Wright 1, Edward O. McFalls 3, Tammy A. Butterick 4,5, Rosemary F. Kelly 1
1Department of Surgery, University of Minnesota, 2School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, 3Cardiology, Richmond VA Medical Center, 4Department of Research Service, Center for Veterans Research and Education, Veterans Affairs Health Care System, 5Department of Neuroscience, University of Minnesota

This study presents a surgical porcine model of chronic myocardial ischemia due to progressive coronary artery stenosis, resulting in impaired cardiac function without infarction. Following ischemia, animals undergo off-pump coronary artery bypass graft with epicardial placement of stem cells-derived exosomes-laden collagen patch. This adjunctive therapy improves myocardial function and recovery.

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Biochemistry

Sample Preparation for In Situ Cryotomography of Mammalian Cells
Noah Weber *1,2, Brennan Hinks *2,3, Jacob Jensen 2, Thomas Lidahl 2,3, Luiza Mendonça 2
1College of Liberal Arts, University of Minnesota, 2Department of Biochemistry, Molecular Biology and Biophysics, Medical School, University of Minnesota, 3College of Biological Sciences, University of Minnesota

This method provides an accessible and flexible protocol for the preparation of electron microscopy (EM) grids for in situ cellular cryotomography and correlative light and electron microscopy (CLEM).

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Medicine

Biobanking of Human Aqueous and Vitreous Liquid Biopsies for Molecular Analyses
Julian Wolf 1,2, Teja Chemudupati 1,2, Aarushi Kumar 1,2, Ditte K. Rasmussen 1,2, Karen M. Wai 2, Robert T. Chang 2, Artis A. Montague 2, Peter H. Tang 3,4, Alexander G. Bassuk 5,6,7, Antoine Dufour 8,9, Prithvi Mruthrunjaya 2, Vinit B. Mahajan 1,2,10
1Molecular Surgery Laboratory, Stanford University, 2Department of Ophthalmology, Byers Eye Institute, Stanford University, 3Department of Ophthalmology and Visual Neurosciences, University of Minnesota, 4Retina Consultants of Minnesota, 5Department of Pediatrics, University of Iowa, 6Department of Neurology, University of Iowa, 7The Iowa Neuroscience Institute (INI), University of Iowa, 8Department of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, 9Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, 10Veterans Affairs Palo Alto Health Care System

This protocol presents an integrated biorepository platform for the standardized collection, annotation, and biobanking of high-quality human aqueous humor and vitreous liquid biopsies for molecular downstream analyses, including proteomics, metabolomics, and glycomics.

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Biology

A Semi-Automated Workflow for the Cryopreservation of Coral Sperm to Support Biobanking and Aquaculture
Jonathan Daly 1,2, Rebecca Hobbs 1, Nikolas Zuchowicz 3, Mary Hagedorn 4,5, Justine K. O’Brien 1,2
1Taronga Institute of Science and Learning, Taronga Conservation Society Australia, 2School of Biological, Earth and Environmental Sciences, University of New South Wales, 3Department of Mechanical Engineering, University of Minnesota, 4Hawaii Institute of Marine Biology, University of Hawaii, 5Smithsonian National Zoo and Conservation Biology Institute

This protocol describes a semi-automated pathway to improve the efficiency and capacity of processing and cryopreservation of sperm from threatened coral species, aiming to secure genetic diversity and support reef restoration efforts.

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Biochemistry

Methodology for Studying Interactions of Vitamin A Membrane Receptors and Opsin Protein with their Ligands in Generating the Retinylidene Protein
Rakesh Radhakrishnan 1, Anjelynt Lor 1, Dorothy Li 1, Deepti Mudaliar 2, Glenn P. Lobo 1
1Department of Ophthalmology and Visual Neurosciences, University of Minnesota, 2High Throughput Screening Laboratory, Institute for Therapeutics Discovery & Development, University of Minnesota

Here, we describe two quantitative methods for studying the protein-ligand interactions of vitamin A membrane receptors and photoreceptor opsin with their respective physiological ligands.

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Developmental Biology

High-resolution Cell Transplantation in Embryonic and Larval Zebrafish
Lindsey S. Qian 1, Rabab Ibrahim 1,2, Adam J. Isabella 1,2
1Department of Genetics, Cell Biology, and Development, University of Minnesota, 2Graduate Program in Molecular, Cellular, Developmental Biology and Genetics, University of Minnesota

Here we present a protocol to transplant cells with high spatial and temporal resolution in zebrafish embryos and larvae at any stage between at least 1 and 7 days post fertilization.

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Quantifying <i>Drosophila melanogaster</i> Eye Phenotypes: A Computational Approach Integrating ilastik and Flynotyper

Quantifying Drosophila melanogaster Eye Phenotypes: A Computational Approach Integrating ilastik and Flynotyper
Qasim Mujteba *1, Madeleine R. Chalmers *1, JiHye Kim 1, Minwoo Baek 1, Nam Chul Kim 1
1Department of Pharmacy Practice and Pharmaceutical Sciences, College of Pharmacy, University of Minnesota

The Drosophila eye system is a useful tool for studying various biological processes, particularly human neurodegenerative diseases. However, manual quantification of rough eye phenotypes can be biased and unreliable. Here we describe a method by which ilastik and Flynotyper are used to quantify eye phenotype in an unbiased way.

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