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University of Illinois at Chicago

61 ARTICLES PUBLISHED IN JoVE

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Biology

Applying Microfluidics to Electrophysiology
David T. Eddington 1
1Dept. of Bioengineering, University of Illinois, Chicago

Applying Microfluidics to Electrophysiology

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Biology

Brain Slice Stimulation Using a Microfluidic Network and Standard Perfusion Chamber
Javeed Shaikh Mohammed 1, Hugo Caicedo 1, Christopher P. Fall 2, David T. Eddington 1
1Dept. of Bioengineering, University of Illinois, Chicago, 2Department of Anatomy and Cell Biology, University of Illinois, Chicago

We demonstrate fabrication of a simple microfluidic device that can be integrated with standard electrophysiology setups to expose microscale surfaces of a brain slice in a well controlled manner to different neurotransmitters.

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Biology

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue
Meirigeng Qi 1, Barbara Barbaro 1, Shusen Wang 1, Yong Wang 1, Mike Hansen 1, Jose Oberholzer 1
1Department of Surgery, University of Illinois, Chicago

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method.

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Biology

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets
Meirigeng Qi 1, Barbara Barbaro 1, Shusen Wang 1, Yong Wang 1, Mike Hansen 1, Jose Oberholzer 1
1Department of Surgery, University of Illinois, Chicago

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.

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Biology

A Multi-Parametric Islet Perifusion System within a Microfluidic Perifusion Device
Adeola F. Adewola 1, Yong Wang 1, Tricia Harvat 1, David T. Eddington 2, Dongyoung Lee 1, Jose Oberholzer 1,2
1Department of Surgery, University of Illinois, Chicago, 2Department of Bioengineering, University of Illinois, Chicago

A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence imaging of calcium influx and mitochondrial potential changes.

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Medicine

Methods to Quantify Pharmacologically Induced Alterations in Motor Function in Human Incomplete SCI
Christopher K. Thompson 1,2, Arun Jayaraman 1, Catherine Kinnaird 1, T. George Hornby 1,3
1Sensory Motor Performance Program, Rehabilitation Institute of Chicago, 2Department of Kinesiology and Nutrition, University of Illinois at Chicago, 3Department of Physical Therapy, University of Illinois at Chicago

This video demonstrates modulation of reflex activity, volitional strength and ambulation through clinical and quantitative assessments in individuals with motor incomplete SCI as a result of acute oral administration of a serotonin reuptake inhibitor (SSRI).

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Biology

Bimolecular Fluorescence Complementation
Katy A. Wong 1, John P. O'Bryan 1
1Department of Pharmacology, University of Illinois at Chicago

The subcellular localization of proteins is important in determining the spatio-temporal regulation of cell signaling. Here, we describe bimolecular fluorescence complementation (BiFC) as a straightforward method for monitoring the spatial interactions of proteins in the cell.

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Bioengineering

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts
Shelby M. King *1, Suzanne Quartuccio *1, Tyvette S. Hilliard *1, Kari Inoue 1, Joanna E. Burdette 1
1Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

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Bioengineering

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness
Myung-Jin Oh 1, Frank Kuhr 1, Fitzroy Byfield 2, Irena Levitan 1
1Section of Respiratory, Critical Care and Sleep Medicine, Department of Medicine, University of Illinois, 2Institute for Medicine and Engineering, University of Pennsylvania

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

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Neuroscience

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells
Erin M. Boisvert 1,2, Kyle Denton 1, Ling Lei 1, Xue-Jun Li 1,3
1Department of Neuroscience, The University of Connecticut Health Center, 2Department of Genetics and Developmental Biology, The University of Connecticut Health Center, 3Stem Cell Institute, The University of Connecticut Health Center

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

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Engineering

Characterization of Electrode Materials for Lithium Ion and Sodium Ion Batteries Using Synchrotron Radiation Techniques
Marca M. Doeff 1, Guoying Chen 1, Jordi Cabana 1,2, Thomas J. Richardson 1, Apurva Mehta 3, Mona Shirpour 1, Hugues Duncan 1, Chunjoong Kim 1, Kinson C. Kam 4, Thomas Conry 5
1Environmental Energy Technologies Division, Lawrence Berkeley National Laboratory, 2Department of Chemistry, University of Illinois at Chicago, 3Stanford Synchrotron Radiation Lightsource, 4Haldor Topsøe A/S, 5PolyPlus Battery Company

We describe the use of synchrotron X-ray absorption spectroscopy (XAS) and X-ray diffraction (XRD) techniques to probe details of intercalation/deintercalation processes in electrode materials for Li-ion and Na-ion batteries. Both in situ and ex situ experiments are used to understand structural behavior relevant to the operation of devices

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Bioengineering

Quantitative and Temporal Control of Oxygen Microenvironment at the Single Islet Level
Joe Fu-Jiou Lo 1, Yong Wang 2,3, Zidong Li 1, Zhengtuo Zhao 1, Di Hu 1, David T. Eddington 3, Jose Oberholzer 2,3
1Department of Mechanical Engineering, University of Michigan-Dearborn, 2Department of Surgery/Transplant, University of Illinois at Chicago, 3Department of Bioengineering, University of Illinois at Chicago

Microfluidic oxygen control confers more than just convenience and speed over hypoxic chambers for biological experiments. Especially when implemented via diffusion through a membrane, microfluidic oxygen can provide simultaneous liquid and gas phase modulations at the microscale-level. This technique enables dynamic multi-parametric experiments critical for studying islet pathophysiology.

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Behavior

Using Eye Movements to Evaluate the Cognitive Processes Involved in Text Comprehension
Gary E. Raney 1, Spencer J. Campbell 1, Joanna C. Bovee 1
1Department of Psychology, University of Illinois at Chicago

The present article describes how to use eye tracking methodologies to study the cognitive processes involved in text comprehension. Descriptions of eye tracking equipment, how to develop experimental stimuli, and procedural recommendations are included. The information presented can be applied to most any study using verbal stimuli.

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Biology

Fluorescence Imaging with One-nanometer Accuracy (FIONA)
Yong Wang *1,2, En Cai *1,2, Janet Sheung 1,2, Sang Hak Lee 1,2, Kai Wen Teng 2,3, Paul R. Selvin 1,2,3
1Department of Physics, University of Illinois at Urbana-Champaign, 2Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, 3Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

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Neuroscience

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy
Kumiko I. Claycomb 1, Kasey M. Johnson 1, Ernesto R. Bongarzone 2, Stephen J. Crocker 1
1Department of Neuroscience, University of Connecticut Health Center, 2Department of Anatomy and Cell Biology, University of Illinois at Chicago

Globoid cells are a defining pathological feature of Krabbe disease, a leukodystrophy currently lacking an effective long-term therapy. We have developed a cell culture model to study the innate biology and pathogenic potential of activated microglia and their transformation into globoid cells.

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Neuroscience

Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals
Shankar Ramachandran 1, Simon Alford 1
1Biological Sciences, University of Illinois at Chicago

Recording Ca2+ currents at the presynaptic release face membrane is key to a precise understanding of Ca2+ entry and neurotransmitter release. We present an acute dissociation of the lamprey spinal cord that yields functional isolated reticulospinal axons, permitting recording directly from the release face membrane of individual presynaptic terminals.

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Biology

In vitro Methylation Assay to Study Protein Arginine Methylation
Rama Kamesh Bikkavilli 1, Sreedevi Avasarala 1, Michelle Van Scoyk 1, Manoj Kumar Karuppusamy Rathinam 1, Jordi Tauler 1, Stanley Borowicz 1,2, Robert A. Winn 1,3
1Department of Pulmonary, Critical Care, Sleep, and Allergy, University of Illinois at Chicago, 2Department of Hematology and Oncology, University of Illinois at Chicago, 3Jesse Brown Veterans Affairs Medical Center

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

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Biology

The Soft Agar Colony Formation Assay
Stanley Borowicz 1, Michelle Van Scoyk 2, Sreedevi Avasarala 2, Manoj Kumar Karuppusamy Rathinam 2, Jordi Tauler 2, Rama Kamesh Bikkavilli 2, Robert A. Winn 2,3
1Department of Hematology and Oncology, University of Illinois at Chicago, 2Department of Pulmonary, Critical Care, Sleep, and Allergy, University of Illinois at Chicago, 3Jesse Brown Veterans Affairs Medical Center

The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

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Neuroscience

Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell
Kelly T. Varga 1, Zhongjiao Jiang 1, Liang-Wei Gong 1
1Department of Biological Sciences, University of Illinois at Chicago

After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

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Immunology and Infection

Pseudomonas aeruginosa Induced Lung Injury Model
Varsha Suresh Kumar 1, Ruxana T. Sadikot 2, Jeanette E. Purcell 3, Asrar B. Malik 1, Yuru Liu 1
1Department of Pharmacology, University of Illinois College of Medicine, University of Illinois at Chicago, 2Section of Pulmonary and Critical Care Medicine, Atlanta VAMC, Emory University, 3Biologic Resources Lab, University of Illinois at Chicago

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

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Neuroscience

A Method of Nodose Ganglia Injection in Sprague-Dawley Rat
Michael W. Calik 1,3, Miodrag Radulovacki 1,2, David W. Carley 1,3
1Center for Narcolepsy, Sleep and Health Research, University of Illinois at Chicago, 2Department of Pharmacology, University of Illinois at Chicago, 3Department of Biobehavioral Health Science, University of Illinois at Chicago

Afferent vagal signaling transmits important information to central nervous system from receptors located in organs of the abdomen and thorax. The nodose ganglia of vagus nerves contain many types of receptors that modulate vagal activity. This protocol describes a method of local injections of neurochemicals into the nodose ganglia.

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JoVE Core

High-definition Fourier Transform Infrared (FT-IR) Spectroscopic Imaging of Human Tissue Sections towards Improving Pathology
Hari Sreedhar *1, Vishal K. Varma *1, Peter L. Nguyen 2, Bennett Davidson 3,4, Sanjeev Akkina 5, Grace Guzman 2, Suman Setty 2, Andre Kajdacsy-Balla 2, Michael J. Walsh 2
1Department of Bioengineering, University of Illinois at Chicago, 2Department of Pathology, University of Illinois at Chicago, 3Department of Biological Sciences, University of Illinois at Chicago, 4Department of Chemistry, University of Illinois at Chicago, 5Department of Nephrology, University of Illinois at Chicago

Fourier Transform Infrared (FT-IR) spectroscopic imaging is a fast and label-free approach to obtain biochemical data sets of cells and tissues. Here, we demonstrate how to obtain high-definition FT-IR images of tissue sections towards improving disease diagnosis.

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Medicine

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis
Giovanni Lugli 1, Yachana Kataria 1, Zachary Richards 1, Peter Gann 1, Xiaofeng Zhou 2, Larisa Nonn 1
1Department of Pathology, University of Illinois at Chicago, 2Department of Periodontics, University of Illinois at Chicago

The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.

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Engineering

High-resolution Thermal Micro-imaging Using Europium Chelate Luminescent Coatings
Timothy M. Benseman 1,2,3, Yang Hao 1,2, Vitalii K. Vlasko-Vlasov 1, Ulrich Welp 1, Alexei E. Koshelev 1, Wai-Kwong Kwok 1, Ralu Divan 4, Courtney Keiser 5, Chiharu Watanabe 6, Kazuo Kadowaki 6
1Materials Science Division, Argonne National Laboratory, 2Department of Physics, University of Illinois at Chicago, 3Department of Physics, CUNY Queens College, 4Center for Nanoscale Materials, Argonne National Laboratory, 5Department of Physics, University of Northern Iowa, 6Institute for Materials Science, University of Tsukuba

Europium thenoyltrifluoroacetonate (EuTFC) has an optical luminescence line at 612 nm, whose activation efficiency decreases strongly with temperature. If a sample coated with a thin film of this material is micro-imaged, the 612 nm luminescent response intensity may be converted into a direct map of sample surface temperature.

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Medicine

A Novel Approach for the Administration of Medications and Fluids in Emergency Scenarios and Settings
Akilesh Honasoge 1, Neal Lyons 2, Kathleen Hesse 3, Braden Parker 3, Robert Mokszycki 2, Kelly Wesselhoff 3, Rolla Sweis 2, Erik B. Kulstad 3
1College of Medicine, University of Illinois at Chicago, 2Department of Pharmacy, Advocate Christ Medical Center, 3Department of Emergency Medicine, Advocate Christ Medical Center

This study presents a novel device that offers an easy route to quickly provide medications and fluids in patients with limited or difficult IV access. This small-diameter device is placed in the distal third of the rectum, allowing for ongoing medication and fluids administration.

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Medicine

A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse
Neda Afsharkhamseh 1, Elham Ghahari 1, Medi Eslani 1, Ali R. Djalilian 1
1Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago

The following article provides an easy, reproducible technique to effectively create a sustainable mouse model of limbal stem cell deficiency (LSCD). This animal model is useful in testing and comparing the efficacy of treatments for limbal stem cell diseases.

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Cancer Research

Synthesis and Characterization of an Aspirin-fumarate Prodrug that Inhibits NFκB Activity and Breast Cancer Stem Cells
Irida Kastrati 1, Loruhama Delgado-Rivera 2, Gergana Georgieva 3, Gregory R. J. Thatcher 2, Jonna Frasor 1
1Physiology and Biophysics, College of Medicine, University of Illinois at Chicago, 2Medicinal Chemistry and Pharmacognosy, College of Medicine, University of Illinois at Chicago, 3College of Pharmacy, University of Illinois at Chicago

This procedure will demonstrate how we synthesized and characterized the anti-NFκB and anti-cancer stem cell activity of an aspirin-fumarate prodrug.

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Biochemistry

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides
Yi-Wen Hsieh 1, Amel Alqadah 1, Chiou-Fen Chuang 1
1Department of Biological Sciences, University of Illinois at Chicago

We describe here an optimized protocol of fluorescent Electrophoretic Mobility Shift Assays (fEMSA) using purified SOX-2 proteins together with infrared fluorescent dye-labeled DNA probes as a case study to tackle an important biological question.

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JoVE Journal

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death
Snezana Vujicic *1,2, Lanfei Feng *1,2, Angelika Antoni 3, Joyce Rauch 4, Jerrold S. Levine 1,2,5
1Section of Nephrology, Department of Medicine, University of Illinois at Chicago, 2Section of Nephrology, Department of Medicine, Jesse Brown Veterans Affairs Medical Center, 3Department of Biology, Kutztown University of Pennsylvania, 4Division of Rheumatology, Department of Medicine, Research Institute of the McGill University Health Centre, 5Department of Microbiology & Immunology, University of Illinois at Chicago

Here, we present a protocol for the determination of intracellular signaling events induced in viable cells by physical interaction with adjacent dead or dying cells. The protocol focuses on signaling events induced by receptor-mediated recognition of the dead cells, as opposed to their phagocytic uptake or release of soluble mediators.

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Biology

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D
Arivarasu N. Anabazhagan *1, Ishita Chatterjee *1, Shubha Priyamvada 1, Anoop Kumar 1, Sangeeta Tyagi 1, Seema Saksena 1,2, Waddah A. Alrefai 1,2, Pradeep K. Dudeja 1,2, Ravinder K. Gill 1
1Department of Medicine, University of Illinois at Chicago, 2Department of Research, Jesse Brown VA Medical Center

We describe simple methods to study the regulation of intestinal serotonin transporter (SERT) function and expression using an in vitro cell culture model of Caco-2 cells grown in 3D and an ex vivo model of mouse intestines. These methods are applicable to the study of other epithelial transporters.

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Medicine

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption
Riya Thomas 1, Kazandra Diaz 1, Kevin P. Koster 1, Leon M. Tai 1
1Department of Anatomy and Cell Biology, University of Illinois at Chicago

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

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Education

Esophageal Heat Transfer for Patient Temperature Control and Targeted Temperature Management
Melissa I. Naiman 1,2, Maria Gray 2, Joseph Haymore 3, Ahmed F. Hegazy 4, Andrej Markota 5, Neeraj Badjatia 6, Erik B. Kulstad 2,7
1Center for Advanced Design, Research, and Exploration, University of Illinois at Chicago, 2Attune Medical, 3University of Maryland School of Nursing, 4University of Western Ontario, 5University Medical Centre Maribor, 6University of Maryland, 7Department of Emergency Medicine, University of Texas, Southwestern Medical Center

This study presents a novel method to provide efficient patient temperature control for cooling or warming patients. A single use, triple lumen device is placed into the esophagus, analogous to a standard orogastric tube, and connects to existing heat exchange units to perform automatic patient temperature management.

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Developmental Biology

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs)
Madeline Williams *1, Smrithi Prem *1, Xiaofeng Zhou 1, Paul Matteson 2, Percy Luk Yeung 3, Chi-Wei Lu 3, Zhiping Pang 4, Linda Brzustowicz 5, James H. Millonig 2, Emanuel Dicicco-Bloom 1
1Department of Neuroscience and Cell Biology, Rutgers Robert Wood Johnson Medical School, 2Center for Advanced Biotechnology and Medicine, Department of Neuroscience and Cell Biology, Rutgers Robert Wood Johnson Medical School, 3The Child Health Institute of NJ, Department of Obstetrics, Gynecology, and Reproductive Services, Rutgers Robert Wood Johnson Medical School, 4The Child Health Institute of NJ, Department of Neuroscience and Cell Biology, Rutgers Robert Wood Johnson Medical School, 5Department of Genetics, Rutgers University

Neurodevelopmental processes such as proliferation, migration, and neurite outgrowth are often perturbed in neuropsychiatric diseases. Thus, we present protocols to rapidly and reproducibly assess these neurodevelopmental processes in human iPSC-derived NPCs. These protocols also allow the assessment of the effects of relevant growth factors and therapeutics on NPC development.

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Bioengineering

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro
Corey M. Rountree 1, John B. Troy 2, Laxman Saggere 1
1Department of Mechanical and Industrial Engineering, University of Illinois at Chicago, 2Department of Biomedical Engineering, Northwestern University

This protocol describes a novel method for investigating a form of chemical neurostimulation of wholemount rat retinas in vitro with the neurotransmitter glutamate. Chemical neurostimulation is a promising alternative to the conventional electrical neurostimulation of retinal neurons for treating irreversible blindness caused by photoreceptor degenerative diseases.

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Bioengineering

Microhoneycomb Monoliths Prepared by the Unidirectional Freeze-drying of Cellulose Nanofiber Based Sols: Method and Extensions
Zheng-Ze Pan 1,2, Hirotomo Nishihara 3, Wei Lv 1, Cong Wang 1,2, Yi Luo 1,2, Liubing Dong 1,2, Houfu Song 1,4, Wenjie Zhang 2, Feiyu Kang 1,2,4, Takashi Kyotani 3, Quan-Hong Yang 1,4,5
1Engineering Laboratory for Functionalized Carbon Materials and Shenzhen Key Laboratory for Graphene-based Materials, Graduate School at Shenzhen, Tsinghua University, 2School of Materials Science and Engineering, Tsinghua University, 3Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 4Tsinghua-Berkeley Shenzhen Institute (TBSI), Tsinghua University, 5School of Chemical Engineering and Technology, Tianjin University

Here, we present a general protocol to prepare a variety of microhoneycomb monoliths (MHMs) in which fluid can pass through with an extremely low pressure drop. MHMs obtained are expected to be used as filters, catalyst supports, flow-type electrodes, sensors and scaffolds for biomaterials.

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Neuroscience

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes
Rafael E. Flores-Obando 1, Mona M. Freidin 2, Charles K. Abrams 2
1Program in Molecular and Cellular Biology, State University of New York Downstate Medical Center, 2Department of Neurology and Rehabilitation, University of Illinois at Chicago

We describe the immunomagnetic isolation of primary mouse oligodendrocytes, which allows the rapid and specific isolation of the cells for in vitro culture.

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Medicine

A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish
Xiao Ma *1,2,3, Yonghe Ding *2,3, Yong Wang 2,3,4, Xiaolei Xu 1,2,3
1Clinical and Translational Sciences Track, Mayo Clinic Graduate School of Biomedical Sciences, 2Department of Biochemistry and Molecular Biology, Mayo Clinic, 3Division of Cardiovascular Diseases, Mayo Clinic, 4Institute of Life Science, Beijing University of Chinese Medicine

A method to generate a doxorubicin-induced cardiomyopathy model in adult zebrafish (Danio rerio) is described here. Two alternative ways of intraperitoneal injection are presented and conditions to reduce variations among different experimental groups are discussed.

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Neuroscience

Quantifying Acute Changes in Renal Sympathetic Nerve Activity in Response to Central Nervous System Manipulations in Anesthetized Rats
Anne M. Fink 1, Caron Dean 2
1Department of Biobehavioral Health Science, College of Nursing, University of Illinois at Chicago, 2Department of Anesthesiology, Medical College of Wisconsin and Zablocki VA Medical Center

Methods for measuring sympathetic and cardiovascular responses to central nervous system (CNS) manipulations are important for advancing neuroscience. This protocol was developed to assist scientists with measuring and quantifying acute changes in renal sympathetic nerve activity (RSNA) in anesthetized rats (non-survival).

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Neuroscience

Laminectomy and Spinal Cord Window Implantation in the Mouse
Elizabeth A. Pietruczyk 1, Terilyn K.L. Stephen 2, Simon Alford 1, Sarah E. Lutz 1
1Department of Anatomy and Cell Biology, University of Illinois at Chicago College of Medicine, 2Medical Scientist Training Program, University of Illinois at Chicago College of Medicine

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

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Bioengineering

Handling and Assessment of Human Primary Prostate Organoid Culture
Tara McCray *1, Zachary Richards *1, Joseph Marsili 1, Gail S. Prins 1,2,3, Larisa Nonn 1,3
1Department of Pathology, University of Illinois at Chicago, 2Departments of Urology, Physiology, and Biophysics, University of Illinois at Chicago, 3University of Illinois Cancer Center

Here, we present a protocol to guide human primary prostate organoid handling then suggest endpoints to assess phenotype. Seeding, culture maintenance, recovery from matrix gel, morphologic quantification, embedding and sectioning, FFPE sectioning, whole-mount staining, and application of commercial assays are described.

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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

Using the Open-Source MALDI TOF-MS IDBac Pipeline for Analysis of Microbial Protein and Specialized Metabolite Data
Chase M. Clark 1, Maria S. Costa 1,2, Erin Conley 1, Emma Li 1, Laura M. Sanchez 1, Brian T. Murphy 1
1Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 2Faculty of Pharmaceutical Sciences, University of Iceland

IDBac is an open-source mass spectrometry-based bioinformatics pipeline that integrates data from both intact protein and specialized metabolite spectra, collected on cell material scraped from bacterial colonies. The pipeline allows researchers to rapidly organize hundreds to thousands of bacterial colonies into putative taxonomic groups, and further differentiate them based on specialized metabolite production.

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Cancer Research

Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry
Katherine E. Zink 1, Matthew Dean 1,2, Joanna E. Burdette 1, Laura M. Sanchez 1
1Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 2Department of Animal Science, University of Illinois at Urbana-Champaign

A novel method of sample preparation was developed to accommodate cell and tissue coculture to detect small molecule exchange using imaging mass spectrometry.

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Immunology and Infection

Electrophysiological Recordings of Single-cell Ion Currents Under Well-defined Shear Stress
Ibra S. Fancher 1, Irena Levitan 1
1Department of Medicine, Division of Pulmonary, Critical Care, Sleep and Allergy, University of Illinois at Chicago

The goal of this protocol is to describe a modified parallel plate flow chamber for use in investigating real time activation of mechanosensitive ion channels by shear stress.

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JoVE Core

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum
Shahin Shabanipour 1,2, Azadeh Dalvand 1,2, Xiaodan Jiao 1,2, Maryam Rahimi Balaei 1,2, Seung H. Chung 3, Jiming Kong 1, Marc. R. Del Bigio 2,4, Hassan Marzban 1,2
1Department of Human Anatomy and Cell Science, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, 2The Children's Hospital Research Institute of Manitoba (CHRIM), Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, 3Department of Oral Biology, University of Illinois at Chicago, 4Department of Pathology, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

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Cancer Research

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures
Wen-Yang Hu 1, Dan-Ping Hu 1, Lishi Xie 1, Lynn A. Birch 1, Gail S. Prins 1,2,3
1Department of Urology, College of Medicine, University of Illinois at Chicago, 2Department of Pathology, College of Medicine, University of Illinois at Chicago, 3University of Illinois Cancer Center, College of Medicine, University of Illinois at Chicago

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

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Developmental Biology

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development
Yong Wang *1, Ding Zhang *2, Fang Zhou 2, Meijun Zhou 2, Qiujie Li 1, Jingyu Chen 3, Jun Yang 1,2
1Department of Physiology and Department of Cardiology of the Second Affiliated Hospital, Zhejiang University School of Medicine, 2Department of Cell Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, 3Wuxi Lung Transplant Center, Wuxi People's Hospital affiliated to Nanjing Medical University

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

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Neuroscience

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia
Yongchao Mou 1,2, Sukhada Mukte 1, Eric Chai 1, Joshua Dein 3, Xue-Jun Li 1,2
1Department of Biomedical Sciences, University of Illinois College of Medicine Rockford, 2Department of Bioengineering, University of Illinois at Chicago, 3MD Program, University of Illinois College of Medicine Rockford

Impaired mitochondrial transport and morphology are involved in various neurodegenerative diseases. The presented protocol uses induced pluripotent stem cell-derived forebrain neurons to assess mitochondrial transport and morphology in hereditary spastic paraplegia. This protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology, which will facilitate the study of neurodegenerative disease.

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Immunology and Infection

Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy
Liangfei Niu 1, Cong Wang 1, Kaile Zhang 1,2, Miaomiao Kang 1,2, Rui Liang 1, Xiaonan Zhang 1, Bo Yan 1
1Shanghai Public Health Clinical Center, Fudan University, 2School of Life Sciences, Bengbu Medical College

This protocol describes a technique for visualizing macrophage behavior and death in embryonic zebrafish during Mycobacterium marinum infection. Steps for the preparation of bacteria, infection of the embryos, and intravital microscopy are included. This technique may be applied to the observation of cellular behavior and death in similar scenarios involving infection or sterile inflammation.

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Biochemistry

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol
Alexandria Slayden 1, Kelsey North 1, Shivantika Bisen 1, Alex M. Dopico 1, Anna N. Bukiya 1, Avia Rosenhouse-Dantsker 2
1Department of Pharmacology, Addiction Science and Toxicology, The University of Tennessee HSC, 2Department of Chemistry, University of Illinois at Chicago

Two methods of cholesterol enrichment are presented: the application of cyclodextrin saturated with cholesterol to enrich mammalian tissues and cells, and the use of cholesterol-enriched phospholipid-based dispersions (liposomes) to enrich Xenopus oocytes. These methods are instrumental for determining the impact of elevated cholesterol levels in molecular, cellular, and organ function.

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Medicine

Taking the Next Step: a Neural Coaptation Orthotopic Hind Limb Transplant Model to Maximize Functional Recovery in Rat
Feibo Zheng 1,3, Andy Tully 1,2, Kyle M Koss 1, Xiaomin Zhang 1, Longhui Qiu 1, Jiao-Jing Wang 1, Bilal A Naved 1,4, David Z Ivancic 1, James M Mathew 1, Jason A Wertheim 1, Zheng Jenny Zhang 1
1Comprehensive Transplant Center and Department of Surgery, Feinberg School of Medicine, Northwestern University, 2Department of Surgery, University of Illinois at Chicago, 3Department of Surgery, Tianjin Occupational Diseases Precaution and Therapeutic Hospital, 4Department of Biomedical Engineering, McCormick School of Engineering, Northwestern University

This protocol presents a robust, reproducible model of vascularized composite allotransplant (VCA) geared toward simultaneous study of immunology and functional recovery. The time invested in meticulous technique in a right mid-thigh hind limb orthotopic transplant with hand sewn vascular anastomoses and neural coaptation yields the ability to study functional recovery.

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Immunology and Infection

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections
Dinesh Jaishankar 1, Cormac Cosgrove 1, Ryan J. Deaton 2, I. Caroline Le Poole 1,3
1Robert Lurie Comprehensive Cancer Center and Department of Dermatology, Feinberg School of Medicine, Northwestern University, 2Department of Pathology, University of Illinois at Chicago, 3Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University

We describe a rapid staining method to perform multispectral imaging on frozen tissues.

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JoVE Core

High-Temperature and High-Pressure In situ Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy
Nicholas R. Jaegers 1, Wenda Hu 2, Yong Wang 1, Jian Zhi Hu 1
1Pacific Northwest National Laboratory, 2Washington State University

The molecular structures and dynamics of solids, liquids, gases, and mixtures are of critical interest to diverse scientific fields. High-temperature, high-pressure in situ MAS NMR enables detection of the chemical environment of constituents in mixed phase systems under tightly controlled chemical environments.

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Immunology and Infection

Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals
Tejabhiram Yadavalli 1, Ipsita Volety 1, Deepak Shukla 1,2
1Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, 2Department of Microbiology and Immunology, University of Illinois at Chicago

We describe the use of a porcine cornea to test the antiviral efficacy of experimental drugs.

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Biochemistry

On-Chip Crystallization and Large-Scale Serial Diffraction at Room Temperature
Linta M. Biju *1, Cong Wang *1, Weijia Kang 1, Irin P. Tom 1, Indika Kumarapperuma 1, Xiaojing Yang 1,2, Zhong Ren 1,3
1Department of Chemistry, University of Illinois at Chicago, 2Department of Ophthalmology and Vision Sciences, University of Illinois at Chicago, 3Renz Research, Inc.

This contribution describes how to set up protein crystallization on crystal-on-crystal devices and how to perform automated serial data collection at room temperature using the on-chip crystallization platform.

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Medicine

Echocardiographic Characterization of Left Ventricular Structure, Function, and Coronary Flow in Neonate Mice
Shamim A. K. Chowdhury 1, Paola C. Rosas 1
1Department of Physiology and Biophysics, Center for Cardiovascular Research, College of Medicine, University of Illinois at Chicago

The present protocol describes the echocardiographic assessment of left ventricular morphology, function, and coronary blood flow in 7-day old neonate mice.

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Biology

Comprehensive Echocardiographic Assessment of Right Ventricle Function in a Rat Model of Pulmonary Arterial Hypertension
Paola C. Rosas *1, Liomar A. A. Neves *2, Peter B. Senese 2, Michael R. Gralinski 2
1Department of Pharmacy Practice, College of Pharmacy, University of Illinois at Chicago, 2CorDynamics, Inc.

The present protocol describes the echocardiographic characterization of right ventricular morphology and function in a rat model of pulmonary arterial hypertension.

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Biology

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications
Thanh Nguyen 1, Sang Joon Ahn 2, Richard West 3, Ibra S. Fancher 1
1Department of Kinesiology and Applied Physiology, College of Health Sciences, University of Delaware, 2Division of Pulmonary, Critical Care, Sleep and Allergy, Department of Medicine, University of Illinois at Chicago, 3University of Delaware Flow Cytometry Core, Delaware Biotechnology Institute, University of Delaware

This protocol details a method for the dissection of mouse adipose depots and the isolation and digestion of respective arteries to liberate and then identify the endothelial cell population. Freshly isolated cells used in downstream applications will advance the understanding of vascular cell biology and the mechanisms of vascular dysfunction.

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Medicine

An Assay to Detect Protection of the Retinal Vasculature from Diabetes-Related Death in Mice
Yanliang Li 1, Andrius Kazlauskas 1,2
1Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, 2Department of Physiology and Biophysics, University of Illinois at Chicago

A protection assay was developed to monitor the retinal vasculature's resilience to death from diabetes/diabetic retinopathy-related insults such as oxidative stress and cytokines.

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Biology

Development and Application of Rapamycin-regulated Tyrosine Phosphatases
Barbara N. Szynal 1, Hanna Bradford-Olson 1, Andrei V. Karginov 1
1Department of Pharmacology and Regenerative Medicine, University of Illinois at Chicago

This protocol describes the design, creation, and application of rapamycin-regulated phosphatases. This method provides high specificity and tight temporal control of phosphatase activation in living cells.

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Biology

Spatiotemporal Control of Protein Activity through Optogenetic Allosteric Regulation
Trisha Bansal 1, Nicholas Lechinsky 1, Andrei V. Karginov 1
1Department of Pharmacology and Regenerative Medicine, University of Illinois at Chicago

This protocol describes the application of an engineered blue-light-activated allosteric switch (LightR) domain for reversible spatiotemporal control of protein activity. Utilizing Src tyrosine kinase as a model, this study offers an elaborate protocol for developing and characterizing light-regulated Src (LightR-Src). It demonstrates the versatility of this approach across enzyme classes.

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