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Stanford University School of Medicine

101 ARTICLES PUBLISHED IN JoVE

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Biology

Orthotopic Mouse Model of Colorectal Cancer
William Tseng 1,2, Xianne Leong 2, Edgar Engleman 2
1Department of Surgery, University of California, San Francisco - UCSF, 2Department of Pathology, Stanford University School of Medicine

Two techniques can be used to establish this model: injection of a cancer cell suspension into the cecal wall or transplantation of a piece of subcutaneous tumor onto the cecum. This model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

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Biology

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells
Kitchener Wilson 1, Jin Yu 1, Andrew Lee 1, Joseph C. Wu 1
1Departments of Radiology and Medicine (Cardiology), Stanford University School of Medicine

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

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Biology

Patch Clamp Recording of Ion Channels Expressed in Xenopus Oocytes
Austin L Brown 1, Brandon E. Johnson 2, Miriam B. Goodman 2
1Department of Molecular and Cellular Physiology, Stanford University , 2Department of Molecular and Cellular Physiology, Stanford University School of Medicine

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

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Biology

Making Patch-pipettes and Sharp Electrodes with a Programmable Puller
Austin L. Brown 1, Brandon E. Johnson 2, Miriam B. Goodman 2
1Department of Molecular and Cellular Physiology, Stanford University , 2Department of Molecular and Cellular Physiology, Stanford University School of Medicine

This video shows how to use a programmable puller to make patch pipettes and sharp electrodes for electrophysiology. The same procedure can be used to make a variety of glass tools, including injection needles.

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Biology

The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia
Hidetoshi Taniguchi 1, Katrin Andreasson 1
1Department of Neurology and Neurological Sciences, Stanford University School of Medicine

The postnatal rat model for hypoxic-ischemic brain injury is a well-established model of human neonatal hypoxic ischemic encephalopathy (HIE). In this article, we describe the model of HIE in post-natal rat pups.

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Biology

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds
Brendan Carvalho 1, David J. Clark 1, David Yeomans 1, Martin S. Angst 1
1Department of Anesthesiology, Stanford University School of Medicine

A technique to collect and measure surgical wound biochemical mediators at specific time points.

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Biology

Pressure-polishing Pipettes for Improved Patch-clamp Recording
Brandon E. Johnson 1, Austin L. Brown 1, Miriam B. Goodman 1
1Department of Molecular and Cellular Physiology, Stanford University School of Medicine

This is a guide to modifying the shape of glass micropipettes. Specifically, by using heat and air pressure the taper is widened without increasing the tip opening, leading to lower pipette resistance. This is critical to obtain low noise recordings of small cells but is useful in many applications.

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Biology

Calcium Imaging of Cortical Neurons using Fura-2 AM
Odmara L Barreto-Chang 1, Ricardo E Dolmetsch 2
1Department of Neurobiology, Stanford University , 2Department of Neurobiology, Stanford University School of Medicine

Calcium signals play a key role in many cellular processes including gene expression, survival and differentiation. Here we demonstrate how to perform calcium imaging using Fura-2 AM. Calcium imaging is a valuable tool to study the regulation of intracellular calcium in real time and its regulation of signaling cascades.

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Biology

Human In-Vivo Bioassay for the Tissue-Specific Measurement of Nociceptive and Inflammatory Mediators
Martin S Angst 1, Martha Tingle 1, Martin Schmelz 2,3, Brendan Carvalho 1, David C Yeomans 1
1Department of Anesthesia, Stanford University School of Medicine, 2Department of Anaesthesiology, University of Mannheim, 3Department of Anaesthesiology, University of Heidelberg

A technique is presented for the in-vivo collection of interstitial fluid samples from pertinent tissue sites (here, experimentally inflamed skin) for the measurement of biochemicals mediating pain and inflammation.

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Biology

Determining heat and mechanical pain threshold in inflamed skin of human subjects
Martin S Angst 1, Martha Tingle 1, Nicholas G Phillips 1, Brendan Carvalho 1
1Department of Anesthesia, Stanford University School of Medicine

Algorithms assessing heat and mechanical pain thresholds in experimentally inflamed skin of human study subjects are shown. The two pain testing paradigms independently examine nociceptive processing by the two major peripheral nerve fiber populations transmitting pain, i.e., non-myelinated C fibers and small myelinated A-delta fibers.

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Biology

Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase
Junya Azuma 1, Tomoko Asagami 2, Ronald Dalman 2, Philip S. Tsao 1
1Department of Cardiovascular Medicine, Stanford University School of Medicine, 2Department of Vascular Surgery, Stanford University School of Medicine

This video shows how to induce abdominal aortic aneurysms (AAA) in mice via transient intraluminal infusion of porcine pancreatic elastase into the infrarenal segment of the abdominal aorta. The model has the ability to add broad insight into the pathobiology of AAA due to the emergence of numerous transgenic and gene knockout mice.

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Biology

Imaging In-Stent Restenosis: An Inexpensive, Reliable, and Rapid Preclinical Model
Tobias Deuse 1, Fumiaki Ikeno 2, Robert C. Robbins 1, Sonja Schrepfer 1
1Department of Cardiothoracic Surgery, Stanford University School of Medicine, 2Stanford University School of Medicine

This video demonstrates how to use a preclinical inexpensive and reliable model to study pathobiological and pathophysiological processes of in-stent restenosis development. Longitudinal in vivo monitoring using OCT (Optical Coherence Tomography) and analysis of OCT images are also demonstrated.

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Biology

Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of Obliterative Airway Disease
Xiaoqin Hua 1, Tobias Deuse 1,2, Karis R. Tang-Quan 1,2,3, Robert C. Robbins 3, Hermann Reichenspurner 1,2, Sonja Schrepfer 1,2,3
1Transplant and Stem Cell Immunobiology Lab (TSI), University Heart Center Hamburg, 2CVRC, University Hospital Hamburg, 3Department of CT Surgery, Stanford University School of Medicine

This video shows and compares two experimental models to study the development of obliterative airway disease (OAD) in mice, the heterotopic and orthotopic tracheal transplantation model.

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Biology

LAD-Ligation: A Murine Model of Myocardial Infarction
Mandy V.V. Kolk 1,2, Danja Meyberg 1,2, Tobias Deuse 1,2, Karis R. Tang-Quan 1,2,3, Robert C. Robbins 3, Hermann Reichenspurner 1,2, Sonja Schrepfer 1,2,3
1Transplant and Stem Cell Immunobiology Lab (TSI), University Heart Center Hamburg, 2CVRC, University Hospital Hamburg, 3Department of CT Surgery, Stanford University School of Medicine

This video demonstrates how to use a fast and reliable model to study pathobiological and pathophysiological processes of myocardial ischemia.

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Medicine

An Experimental Paradigm for the Prediction of Post-Operative Pain (PPOP)
Ruth Landau 1, John C. Kraft 1, Lisa Y. Flint 1, Brendan Carvalho 1, Philippe Richebé 1, Monica Cardoso 1, Patricia Lavand'homme 1, Michal Granot 1, David Yarnitsky 1, Alex Cahana 1
1Department of Anesthesiology and Pain Medicine, University of Washington School of Medicine

Diffuse noxious inhibitory control, temporal summation and wound hyperalgesia testing are demonstrated in the obstetric patient. These tests evaluate inhibitory and excitatory mechanisms of pain processing and are here utilized to evaluate endogenous analgesia at different time-points during pregnancy and the peripartum period to help reveal individual s risk for persistent pain.

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Medicine

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy
Mandy Stubbendorff 1, Tobias Deuse 1,2, Anna Hammel 1, Robert C. Robbins 2, Hermann Reichenspurner 1, Sonja Schrepfer 1,2
1University Heart Center Hamburg, Transplant and Stem Cell Immunobiology Lab (TSI), University Hospital Hamburg, 2Stanford University School of Medicine

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

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Immunology and Infection

Transurethral Induction of Mouse Urinary Tract Infection
Kim H. Thai 1, Anuradha Thathireddy 2, Michael H. Hsieh 2
1Earth Systems Program, School of Earth Sciences, Stanford University , 2Department of Urology, Stanford University School of Medicine

This video will demonstrate methods to transurethrally induce mouse urinary tract infections and quantify the extent of resulting infections.

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Neuroscience

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury
Qian Wang 1, Katrin Andreasson 1
1Department of Neurology and Neurological Sciences, Stanford University School of Medicine

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

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Medicine

Dissection of Human Vitreous Body Elements for Proteomic Analysis
Jessica M. Skeie 1, Vinit B. Mahajan 1
1Department of Ophthalmology and Visual Sciences, Omics Laboratory, University of Iowa

This video shows an effective technique for differentiating and dissecting the various semi-transparent structures of the human vitreous body in post mortem eyes.

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Medicine

Mouse Bladder Wall Injection
Chi-Ling Fu 1, Charity A. Apelo *1, Baldemar Torres *1, Kim H. Thai 1, Michael H. Hsieh 1
1Department of Urology, Stanford University School of Medicine

Mouse bladder wall injection is a useful approach to orthotopically study bladder stem cell and cancer biology. This delicate microsurgical method can be mastered with careful technique and practice.

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Bioengineering

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)
Lenard Conradi 1,2, Christiane Pahrmann 1, Stephanie Schmidt 1, Tobias Deuse 1,3, Arne Hansen 2, Alexandra Eder 2, Hermann Reichenspurner 1, Robert C. Robbins 3, Thomas Eschenhagen 2, Sonja Schrepfer 1,3
1Transplant and Stem Cell Immunobiology Lab (TSI) and CVRC, University Hospital Hamburg, University Heart Center Hamburg, 2Department of Experimental and Clinical Pharmacology and Toxicology, University Heart Center Hamburg, 3CT Surgery, Stanford University School of Medicine

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

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Biology

Associated Chromosome Trap for Identifying Long-range DNA Interactions
Jianqun Ling 1, Andrew R. Hoffman 1
1Medical Service, VA Palo Alto Health Care System , Stanford University School of Medicine

The associated chromosome trap (ACT) assay is a novel unbiased method for identifying long-range DNA interactions. The characterization of long range DNA interactions will allow us to determine the relationship of nuclear architecture to gene expression in both normal physiology and in diseased states.

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Biology

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses
Jessica M. Skeie 1,2, Stephen H. Tsang 3, Vinit B. Mahajan 1,2
1Omics Laboratory, University of Iowa, 2Ophthalmology and Visual Sciences, University of Iowa, 3Harkness Eye Institute, Columbia University College of Physicians and Surgeons

The dissection technique illustrates evisceration of the vitreous, retina, and lens from the mouse eye, separation by centrifugation, and characterization with protein assays.

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Medicine

Mouse Eye Enucleation for Remote High-throughput Phenotyping
Vinit B. Mahajan 1,2, Jessica M. Skeie 1,2, Amir H. Assefnia 2,3, MaryAnn Mahajan 1,2, Stephen H. Tsang 2,4
1Department of Ophthalmology and Visual Sciences, University of Iowa, 2Omics Laboratory, University of Iowa, 3School of Dentistry, UCLA, 4Bernard and Shirlee Brown Glaucoma Laboratory, Department of Ophthalmology, College of Physicians and Surgeons, Columbia University

The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens.

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Neuroscience

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry
Taha A. Jan 1, Renjie Chai 1, Zahra N. Sayyid 1, Alan G. Cheng 1
1Department of Otolaryngology-Head and Neck Surgery, Stanford University School of Medicine

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of β-galactosidase prior to separation via flow cytometry.

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Neuroscience

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones
Andrew J. Shepherd 1, Durga P. Mohapatra 1,2
1Department of Pharmacology, The University of Iowa, 2Department of Anesthesia, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

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Medicine

Human Internal Mammary Artery (IMA) Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis
Xiaoqin Hua 1,2, Tobias Deuse 1,2, Evangelos D. Michelakis 3, Alois Haromy 3, Phil S. Tsao 4, Lars Maegdefessel 4, Reinhold G. Erben 5, Claudia Bergow 5, Boris B. Behnisch 6, Hermann Reichenspurner 1,2, Robert C. Robbins 7, Sonja Schrepfer 1,2,7
1University Heart Center Hamburg, TSI-Lab, Germany, 2Cardiovascular Research Center, University of Hamburg, 3Department of Medicine, Cardiology Division, Pulmonary Hypertension Program, University of Alberta, 4Department of Medicine, Stanford University School of Medicine , 5Department of Biomedical Sciences, Institute of Physiology, Pathophysiology, and Biophysics, University of Veterinary Medicine, Vienna, 6Translumina GmbH, Hechingen, 7Department of Cardiothoracic Surgery, Stanford University School of Medicine

This video shows a model to study the development of intimal hyperplasia after stent deployment using a human vessel (IMA) in an immunodeficient rat model.

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Biology

Chromatin Isolation by RNA Purification (ChIRP)
Ci Chu 1, Jeffrey Quinn 1, Howard Y. Chang 1
1Howard Hughes Medical Institute and Program in Epithelial Biology, Stanford University School of Medicine

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

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Medicine

Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate
David D. Lo *1,2, Jeong S. Hyun *1,3, Michael T. Chung 1, Daniel T. Montoro 1, Andrew Zimmermann 1, Monica M. Grova 1,4, Min Lee 5, Derrick C. Wan 1, Michael T. Longaker 1
1Department of Surgery, Stanford University , 2Department of Surgery, Duke University , 3Department of Surgery, Saint Joseph Mercy Hospital, 4School of Medicine, University of California, San Francisco , 5School of Dentistry, University of California, Los Angeles

This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.

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Medicine

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye
Katherine J. Wert 1,2, Jessica M. Skeie 3,4, Richard J. Davis 1, Stephen H. Tsang 1,3, Vinit B. Mahajan 3,4
1Bernard and Shirlee Brown Glaucoma Laboratory, Department of Ophthalmology, Columbia University , 2Institute of Human Nutrition, College of Physicians & Surgeons, Columbia University , 3Omics Laboratory, University of Iowa , 4Department of Ophthalmology and Visual Sciences, University of Iowa

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

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Medicine

A Contusion Model of Severe Spinal Cord Injury in Rats
Vibhor Krishna 1, Hampton Andrews 1, Xing Jin 2, Jin Yu 1, Abhay Varma 1, Xuejun Wen 3, Mark Kindy 1
1Department of Neuroscience, Division of Neurosurgery, Medical University of South Carolina, 2Bioengineering, Clemson University, 3Clemson-MUSC Bioengineering Joint Program

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

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Medicine

Collecting And Measuring Wound Exudate Biochemical Mediators In Surgical Wounds
Brendan Carvalho 1, David J Clark 1, David Yeomans 1, Martin S Angst 1
1Department of Anesthesia, Stanford University School of Medicine

This article provides a detailed and visual description of a methodology for collecting and measuring biochemical inflammatory and nociceptive mediators at the surgical wound site following cesarean delivery. This human bioassay has been used to determine correlations between wound and serum cytokine concentrations and drug-mediated changes in wound cytokines, chemokines and neuropetides.

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Medicine

Parabiosis in Mice: A Detailed Protocol
Paniz Kamran 1,2, Konstantina-Ioanna Sereti 1,2, Peng Zhao 1,2, Shah R. Ali 3, Irving L. Weissman 3, Reza Ardehali 1,2
1Department of Medicine-Division of Cardiology, University of California, Los Angeles, 2Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, 3Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine

Parabiotic joining of two organisms leads to the development of a shared circulatory system. In this protocol, we describe the surgical steps to form a parabiotic connection between a wild-type mouse and a constitutive GFP-expressing mouse.

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Chemistry

Isolation and Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography
Samantha M. Desmarais 1, Felipe Cava 2, Miguel A. de Pedro 3, Kerwyn Casey Huang 1,4
1Department of Bioengineering, Stanford University, 2Department of Molecular Biology and Laboratory for Molecular Infection Medicine Sweden, Umeå Centre for Microbial Research, Umeå University, 3Campus de Cantoblanco, Universidad Autonoma de Madrid, 4Department of Microbiology and Immunology, Stanford University School of Medicine

The bacterial cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by peptides. Ultra Performance Liquid Chromatography provides high resolution and throughput for novel discoveries of peptidoglycan composition. We present a procedure for the isolation of cell walls (sacculi) and their subsequent preparation for analysis via UPLC.

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Engineering

Laser-induced Breakdown Spectroscopy: A New Approach for Nanoparticle's Mapping and Quantification in Organ Tissue
Lucie Sancey 1, Vincent Motto-Ros 2, Shady Kotb 1, Xiaochun Wang 3, François Lux 1, Gérard Panczer 3, Jin Yu 2, Olivier Tillement 1
1ILM-FENNEC UMR 5306, CNRS - Université Lyon 1, 2ILM-PUBLI UMR 5306, CNRS - Université Lyon 1, 3ILM-SOPRANO UMR 5306, CNRS - Université Lyon 1

Laser-induced breakdown spectroscopy performed on thin organ and tumor tissue successfully detected natural elements and artificially injected gadolinium (Gd), issued from Gd-based nanoparticles. Images of chemical elements reached a resolution of 100 μm and quantitative sub-mM sensitivity. The compatibility of the setup with standard optical microscopy emphasizes its potential to provide multiple images of a same biological tissue.

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Medicine

Inducing Myointimal Hyperplasia Versus Atherosclerosis in Mice: An Introduction of Two Valid Models
Mandy Stubbendorff *1,2, Xiaoqin Hua *1,2, Tobias Deuse 1,2,3, Ziad Ali 4,5, Hermann Reichenspurner 2,3, Lars Maegdefessel 6, Robert C. Robbins 7, Sonja Schrepfer 1,2,3,4
1Transplant and Stem Cell Immunobiology Lab, Cardiovascular Research Center, University Hospital Hamburg, 2Cardiovascular Research Center (CVRC) and DZHK University Hamburg, 3Department of Cardiovascular Surgery, University Heart Center Hamburg, 4Center for Interventional Vascular Therapy, Division of Cardiology, Columbia University, 5Cardiovascular Research Foundation, New York, 6Karolinska Institute, Stockholm, 7Department of Cardiothoracic Surgery, Stanford University School of Medicine, Falk Cardiovascular Research Center

This video shows two models of intimal plaque development in murine arteries and emphasizes the differences in myointimal hyperplasia and atherosclerosis.

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Neuroscience

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells
Pengbo Zhang *1, Ninuo Xia *1, Renee A. Reijo Pera 1,2
1Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, 2Department of Obstetrics and Gynecology, Stanford University School of Medicine

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson’s disease.

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Biology

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry
Subarna Bhattacharya *1, Paul W. Burridge *2, Erin M. Kropp 1, Sandra L. Chuppa 1, Wai-Meng Kwok 3, Joseph C. Wu 2, Kenneth R. Boheler 4,5, Rebekah L. Gundry 1,6
1Department of Biochemistry, Medical College of Wisconsin, 2Stanford Cardiovascular Institute, Stanford University School of Medicine, 3Department of Anesthesiology, Medical College of Wisconsin, 4Stem Cell and Regenerative Medicine Consortium, LKS Faculty of Medicine, Hong Kong University, 5Division of Cardiology, Johns Hopkins University School of Medicine, 6Cardiovascular Research Center, Biotechnology and Bioengineering Center, Medical College of Wisconsin

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

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JoVE Journal

Osteoclast Derivation from Mouse Bone Marrow
Ruth Tevlin *1, Adrian McArdle *1,2, Charles K.F. Chan 2, John Pluvinage 2, Graham G. Walmsley 1,2, Taylor Wearda 1,2, Owen Marecic 1,2, Michael S. Hu 1, Kevin J. Paik 1, Kshemendra Senarath-Yapa 1, David A. Atashroo 1, Elizabeth R. Zielins 1, Derrick C. Wan 1, Irving L. Weissman 1,2, Michael T. Longaker 1,2
1Hagey Laboratory for Pediatric Regenerative Medicine, Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University School of Medicine, 2Institute for Stem Cell Biology and Regenerative Medicine, Stanford University

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

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Developmental Biology

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis
Elizabeth R. Zielins *1, Ruth Tevlin *1, Michael S. Hu 1, Michael T. Chung 1, Adrian McArdle 1, Kevin J. Paik 1, David Atashroo 1, Christopher R. Duldulao 1, Anna Luan 1, Kshemendra Senarath-Yapa 1, Graham G. Walmsley 1, Taylor Wearda 1, Michael T. Longaker 1,2, Derrick C. Wan 1
1Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, 2Institute for Stem Cell Biology and Regenerative Medicine, Stanford University

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

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Medicine

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography
David A. Atashroo *1, Kevin J. Paik *1, Michael T. Chung 1, Adrian McArdle 1, Kshemendra Senarath-Yapa 1, Elizabeth R. Zielins 1, Ruth Tevlin 1, Christopher R. Duldulao 1, Graham G. Walmsley 1, Taylor Wearda 1, Owen Marecic 1, Michael T. Longaker 1,2, Derrick C. Wan 1,2
1Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Plastic and Reconstructive Surgery Division, Stanford University School of Medicine, 2Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

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Medicine

A Mouse Fetal Skin Model of Scarless Wound Repair
Graham G. Walmsley *1,2, Michael S. Hu *1,2,3, Wan Xing Hong 1,4, Zeshaan N. Maan 1, H. Peter Lorenz 1, Michael T. Longaker 1,2
1Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, 2Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, 3Department of Surgery, John A. Burns School of Medicine, University of Hawai'i, 4University of Central Florida College of Medicine

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

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Neuroscience

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice
Devsmita Das 1,2, Cristy Phillips 3, Bill Lin 1, Fatemeh Mojabi 1, Mehmet Akif Baktir 2, Van Dang 1,2, Ravikumar Ponnusamy 1, Ahmad Salehi 1,2
1VA Palo Alto Health Care System, 2Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, 3Department of Physical Therapy, Arkansas State University

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

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Developmental Biology

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation
Arun Sharma *1, Guang Li *1, Kuppusamy Rajarajan *1, Ryoko Hamaguchi 1, Paul W. Burridge 1, Sean M. Wu 1,2
1Stanford Cardiovascular Institute, Stanford University School of Medicine, 2Institute of Stem Cell Biology and Regenerative Medicine, Cardiovascular Medicine Division, Department of Medicine, Child Health Research Institute, Stanford University School of Medicine

Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening.

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Biology

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle
Jared M. Schrader 1, Lucy Shapiro 1
1Department of Developmental Biology, Stanford University School of Medicine

Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.

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Medicine

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche
Zachary S. Templeton 1, Michael H. Bachmann 1, Rajiv V. Alluri 1, William J. Maloney 2, Christopher H. Contag 1, Bonnie L. King 1
1Department of Pediatrics, Stanford University School of Medicine, 2Department of Orthopaedic Surgery, Stanford University School of Medicine

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

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Medicine

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model
Russell K. Pachynski 1, Alexander Scholz 3, Justin Monnier 2,3, Eugene C. Butcher 3, Brian A. Zabel 2
1Division of Oncology, Department of Medicine, Washington University School of Medicine, 2Palo Alto Institute for Research and Education, Veterans Affairs Palo Alto Health Care System, 3Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

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Immunology and Infection

Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice
Nicolas Gaudenzio 1, Riccardo Sibilano 1, Philipp Starkl 1, Mindy Tsai 1, Stephen J. Galli 1,2, Laurent L. Reber 1
1Department of Pathology, Stanford University School of Medicine, 2Department of Microbiology & Immunology, Stanford University School of Medicine

We describe a method for the generation of in vitro derived mast cells, their engraftment into mast cell-deficient mice, and the analysis of the phenotype, numbers and distribution of engrafted mast cells at different anatomical sites. This protocol can be used to assess the functions of mast cells in vivo.

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Developmental Biology

Murine Dermal Fibroblast Isolation by FACS
Graham G. Walmsley *1,2, Zeshaan N. Maan *1, Michael S. Hu *1,2,3, David A. Atashroo 1, Alexander J. Whittam 1, Dominik Duscher 1, Ruth Tevlin 1, Owen Marecic 1, H. Peter Lorenz 1, Geoffrey C. Gurtner 1, Michael T. Longaker 1,2
1Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, 2Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, 3Department of Surgery, John A. Burns School of Medicine, University of Hawai'i

Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

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Chemistry

Development of a Backbone Cyclic Peptide Library as Potential Antiparasitic Therapeutics Using Microwave Irradiation
Nir Qvit 1, Opher S. Kornfeld 1
1Department of Chemical and Systems Biology, Stanford University School of Medicine

A simple and general method for the synthesis of cyclic peptides using microwave irradiation is outlined. This procedure enables the synthesis of backbone cyclic peptides with a collection of different conformations while retaining the side chains and the pharmacophoric moieties., and therefore, allows to screen for the bioactive conformation.

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Medicine

A High-content In Vitro Pancreatic Islet β-cell Replication Discovery Platform
Zhengshan Zhao 1, Yassan Abdolazimi 1, Neali A. Armstrong 1, Justin P. Annes 1
1Department of Medicine, Division of Endocrinology, Stanford University School of Medicine

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet β-cell replication and to develop methods for stimulating β-cell regeneration. Herein a high-content screening method to identify and assess the β-cell replication-promoting activity of small molecules is presented.

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Medicine

Creation of Abdominal Adhesions in Mice
Clement D. Marshall 1, Michael S. Hu 1, Tripp Leavitt 1, Leandra A. Barnes 1, Alexander T.M. Cheung 1, Samir Malhotra 1, H. Peter Lorenz 1, Michael T. Longaker 1
1Hagey Laboratory for Pediatric Regenerative Medicine, Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University School of Medicine

Abdominal adhesions that form after surgery are a major cause of pain, infertility, and hospitalization and reoperation for small bowel obstruction. Our surgical procedure for creating abdominal adhesions in mice is a reliable tool to study the mechanisms underlying the formation of adhesions.

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Neuroscience

Dynamic Quantitative Sensory Testing to Characterize Central Pain Processing
Ian G. Mackey 1, Eric A. Dixon 1, Kevin Johnson 1, Jiang-Ti Kong 1
1Anesthesiology, Perioperative and Pain Medicine, Stanford University School of Medicine

By assessing pain in response to repetitive or different types of standardized stimuli, dynamic quantitative sensory testing (QST) can reveal changes in the central processing of pain. We present methods to optimize and individualize two dynamic QST measures: temporal summation (TS) and conditioned pain modulation (CPM).

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Medicine

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency
Dong Wang 1,2,3,4, Grigol Tediashvili 1,2,3, Simon Pecha 4, Hermann Reichenspurner 4, Tobias Deuse 1,2,3,4, Sonja Schrepfer 1,2,3,4
1Transplant and Stem Cell Immunobiology Lab, University Heart Center Hamburg, 2Department of Surgery, Transplant and Stem Cell Immunobiology Lab, University of California San Francisco (UCSF), 3Cardiovascular Research Center (CVRC) and DZHK German Center for Cardiovascular Research), partner site Hamburg/Kiel/Luebeck, 4Cardiovascular Surgery, University Heart Center Hamburg

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

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Developmental Biology

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model
Clement D. Marshall 1, Elizabeth R. Zielins 1, Elizabeth A. Brett 1, Charles P. Blackshear 1, Michael S. Hu 1, Tripp Leavitt 1, Leandra A. Barnes 1, H. Peter Lorenz 1, Michael T. Longaker 1, Derrick C. Wan 1
1Hagey Laboratory for Pediatric Regenerative Medicine, Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University School of Medicine

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

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Cancer Research

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)
Grant L. Lin 1, Michelle Monje 2
1Graduate Program in Neuroscience, Department of Neurology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, 2Departments of Neurology, Neurosurgery, Pathology and Pediatrics, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine

This protocol describes a method for the rapid processing of post-mortem diffuse intrinsic pontine glioma samples for the establishment of patient-derived cell culture models or direct characterization of tumor and microenvironmental cells.

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Biology

High-throughput Screening for Protein-based Inheritance in S. cerevisiae
James S. Byers 1, Daniel F. Jarosz 1,2
1Department of Developmental Biology, Stanford University School of Medicine, 2Department of Chemical and Systems Biology, Stanford University School of Medicine

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.

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Biochemistry

Dissection of Human Retina and RPE-Choroid for Proteomic Analysis
Thiago Cabral *1,2,7,8, Marcus A. Toral *3,4, Gabriel Velez 3,4, James E. DiCarlo 1,2, Anuradha M. Gore 3, MaryAnn Mahajan 3, Stephen H. Tsang 1,2, Alexander G. Bassuk 5,6, Vinit B. Mahajan 3,9
1Barbara & Donald Jonas Stem Cell Laboratory, and Bernard & Shirlee Brown Glaucoma Laboratory, Department of Pathology & Cell Biology, Institute of Human Nutrition, College of Physicians and Surgeons, Columbia University, 2Edward S. Harkness Eye Institute, New York-Presbyterian Hospital, 3Omics Laboratory, Byers Eye Institute, Department of Ophthalmology, Stanford University, 4Medical Scientist Training Program, University of Iowa, 5Department of Pediatrics, University of Iowa, 6Department of Neurology, University of Iowa, 7Department of Ophthalmology, Federal University of Sao Paulo (UNIFESP), 8Department of Ophthalmology, Federal University of EspÍrito Santo (UFES), 9Palo Alto Veterans Administration, Palo Alto, CA

The human retina is composed of functionally and molecularly distinct regions, including the fovea, macula, and peripheral retina. Here, we describe a method using punch biopsies and manual removal of tissue layers from a human eye to dissect and collect these distinct retinal regions for downstream proteomic analysis.

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Medicine

Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta
Grigol Tediashvili 1,2,3, Dong Wang 1,2,3,4, Hermann Reichenspurner 4, Tobias Deuse 1,2,3,4, Sonja Schrepfer 1,2,3,4
1Transplant and Stem Cell Immunobiology Lab, University Heart Center, 2Department of Surgery, Transplant and Stem Cell Immunobiology Lab, University of California San Francisco (UCSF), 3Cardiovascular Research Center (CVRC) and DZHK German Center for Cardiovascular Research, 4Cardiovascular Surgery, University Heart Center

This article demonstrates a murine model to study the development of myointimal hyperplasia (MH) after aortic balloon injury.

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Neuroscience

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans
Holger Fehlauer *1, Adam L. Nekimken *1,2, Anna A. Kim 1,2, Beth L. Pruitt 1,2,3, Miriam B. Goodman 1,2, Michael Krieg 4
1Department of Molecular and Cellular Physiology, Stanford University, 2Department of Mechanical Engineering, Stanford University, 3Department of Bioengineering, Stanford University, 4Group of Neurophotonics and Mechanical Systems Biology, The Institute of Photonic Sciences (ICFO)

New tools for mechanobiology research are needed to understand how mechanical stress activates biochemical pathways and elicits biological responses. Here, we showcase a new method for selective mechanical stimulation of immobilized animals with a microfluidic trap allowing high-resolution imaging of cellular responses.

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Biology

Intracavernosal Pressure Recording to Evaluate Erectile Function in Rodents
Feng Pan *2, Jie Zhang *3, Yuyan Liu 1, Liangsheng Lu 1, Xuefeng Qiu 4, Kangtai Lv 5, Qipeng Zhang 1
1State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Jiangsu Engineering Research Center for microRNA Biology and Biotechnology, Nanjing University, 2Department of Andrology, The Affiliated Obstetrics and Gynecology Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital, 3School of Pharmaceutical Sciences, Nanjing Tech University, 4Department of Urology, Affiliated Drum Tower Hospital, School of Medicine, Nanjing University, 5Department of Ultrasound, Nanjing Qixia District Maternity and Child Health Care Hospital

Intracavernosal pressure recording (ICP) is an important method to evaluate the erectile function of experimental animals. Here, a detailed protocol is demonstrated for the recording procedure of ICP by catheterizing the crura penis and then electrically stimulating the cavernous nerves in rats.

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Engineering

An Available Technique for Preparation of New Cast MnCuNiFeZnAl Alloy with Superior Damping Capacity and High Service Temperature
Dong Li 1,3, Wenbo Liu 1,2, Ning Li 1, Zhenyu Zhong 1, Jiazhen Yan 1, Sanqiang Shi 2
1School of Manufacturing Science and Engineering, Sichuan University, 2Department of Mechanical Engineering, Hong Kong Polytechnic University, 3Department of Mechanical and Electrical Engineering, Chengdu Aeronautic Polytechnic

Here we present a protocol to obtain a novel Mn-Cu-based alloy with excellent comprehensive performances by a high-quality smelting technology and reasonable heat treatment methods.

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Developmental Biology

Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration
Jonathan M. Tsai 1,2, Irving L Weissman 1,2, Yuval Rinkevich 3,4
1Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, 2Department of Developmental Biology, Stanford University School of Medicine, 3Comprehensive Pneumology Center, Institute of Lung Biology and Disease, Helmholtz Zentrum München, 4German Center for Lung Research (DZL)

Here, we present a new method for partial resection of the left hepatic lobe in neonatal (day 0) mice. This new protocol is suitable for studying acute liver injury and injury response in the neonatal setting.

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Immunology and Infection

A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells
Effie E Bastounis 1, Fabian E Ortega 1, Ricardo Serrano 2, Julie A Theriot 3
1Department of Biochemistry, Stanford University School of Medicine, 2Department of Mechanical and Aerospace Engineering, University of California San Diego, 3Departments of Biochemistry, Microbiology and Immunology and Howard Hughes Medical Institute, Stanford University School of Medicine

We have developed a multi-well format polyacrylamide-based assay for probing the effect of extracellular matrix stiffness on bacterial infection of adherent cells. This assay is compatible with flow cytometry, immunostaining, and traction force microscopy, allowing for quantitative measurements of the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria.

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Bioengineering

Electrically Conductive Scaffold to Modulate and Deliver Stem Cells
Byeongtaek Oh 1, Alexa Levinson 1, Vivek Lam 1, Shang Song 1, Paul George 1,2
1Department of Neurology and Neurological Sciences, Stanford University School of Medicine, 2Stanford Stroke Center and Stanford University School of Medicine

This protocol describes fabrication of a cell culture system to allow seeding of stem cells on a conductive polymer scaffold for in vitro electrical stimulation and subsequent in vivo implantation of the stem cell-seeded scaffold using a minimally invasive technique.

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Education

CRISPR-Mediated Reorganization of Chromatin Loop Structure
Stefanie L. Morgan *1,2, Erin Y. Chang *1, Natasha C. Mariano 1, Abel Bermudez 3, Nicole L. Arruda 4, Fanting Wu 5, Yunhai Luo 1, Gautam Shankar 1, Star K. Huynh 1, Chiao-Chain Huang 5, Sharon J. Pitteri 3, Kevin C. Wang 1,2,6
1Department of Dermatology, Program in Epithelial Biology, Stanford University School of Medicine, 2Program in Cancer Biology, Stanford University School of Medicine, 3Canary Center for Cancer Early Detection, Department of Radiology, Stanford University School of Medicine, 4Department of Biology, Bridgewater State University, 5System Biosciences, 6Veterans Affairs Healthcare System

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

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JoVE Journal

Imaging FITC-dextran as a Reporter for Regulated Exocytosis
Ofir Klein 1, Amit Roded 1, Koret Hirschberg 2, Mitsunori Fukuda 3, Stephen J. Galli 4, Ronit Sagi-Eisenberg 1
1Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, 2Department of Pathology, Sackler Faculty of Medicine, Tel Aviv University, 3Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, 4Departments of Pathology and of Microbiology and Immunology and Sean N. Parker Center for Allergy and Asthma Research, School of Medicine, Stanford University

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

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Medicine

Targeted and Selective Treatment of Pluripotent Stem Cell-derived Teratomas Using External Beam Radiation in a Small-animal Model
Karim Sallam 1,2,3, June-Wha Rhee 1,2, Tony Chour 1, Jessica D'addabbo 1,2,3, Andrew S. Lee 1,2,4,5,6, Edward Graves 4,5,7, Patricia K. Nguyen 1,2,3
1Stanford Cardiovascular Institute, Stanford University School of Medicine, 2Department of Medicine, Division of Cardiology, Stanford University School of Medicine, 3Medical Service, Cardiology Section, Veteran Affairs Palo Alto Health Care System, 4Department of Pathology, Stanford University School of Medicine, 5Department of Radiology, Molecular Imaging Program, Stanford University School of Medicine, 6Peking University Shenzhen Health Science Institute, 7Department of Radiation Oncology, Stanford University School of Medicine

Research on treatment strategies for pluripotent stem cell-derived teratomas is important for the clinical translation of stem cell therapy. Here, we describe a protocol to, first, generate stem cell-derived teratomas in mice and, then, to selectively target and treat these tumors in vivo using a small-animal irradiator.

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Biochemistry

How to Quantify the Fraction of Photoactivated Fluorescent Proteins in Bulk and in Live Cells
Vanessa Chen 1, Malte Renz 1
1Gynecologic Oncology Division, Stanford University School of Medicine

Here, we present a protocol that involves genetically coupled spectrally distinct photoactivatable and fluorescent proteins. These fluorescent protein chimeras permit quantification of the PA-FP fraction that is photoactivated to be fluorescent, i.e., the photoactivation efficiency. The protocol reveals that different modes of photoactivation yield different photoactivation efficiencies.

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Developmental Biology

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells
Kyle M. Loh 1,2, Amrita Palaria 1, Lay Teng Ang 1
1Institute for Stem Cell Biology & Regenerative Medicine, Stanford-UC Berkeley Siebel Stem Cell Institute, Stanford University School of Medicine, 2Department of Developmental Biology, Stanford University School of Medicine

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

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Developmental Biology

Transient Treatment of Human Pluripotent Stem Cells with DMSO to Promote Differentiation
Danielle Sambo 1, Jingling Li 1, Thomas Brickler 1, Sundari Chetty 1,2
1Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, 2Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine

Generating differentiated cell types from human pluripotent stem cells (hPSCs) holds great therapeutic promise but remains challenging. PSCs often exhibit an inherent inability to differentiate even when stimulated with a proper set of signals. Described here is a simple tool to enhance multilineage differentiation across a variety of PSC lines.

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Medicine

A Cryoinjury Model to Study Myocardial Infarction in the Mouse
Dong Wang *1,2, Grigol Tediashvili *1,2, Xiaomeng Hu 1,2, Alessia Gravina 2, Sivan G. Marcus 1,2, Hao Zhang 4, Jeffrey E Olgin 4, Tobias Deuse 1,2,5, Sonja Schrepfer 1,2,3,5
1Transplant and Stem Cell Immunobiology Lab, University Heart Center, 2Department of Surgery, Transplant and Stem Cell Immunobiology Lab, University of California San Francisco, 3Cardiovascular Research Center (CVRC) and DZHK German Center for Cardiovascular Research, 4Division of Cardiology, Cardiovascular Research Institute, University of California San Francisco, 5Cardiovascular Surgery, University Heart Center

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

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Medicine

Immunoglobulin G N-Glycan Analysis by Ultra-Performance Liquid Chromatography
Di Liu *1, Xizhu Xu *2, Yuejin Li 2, Jie Zhang 1, Xiaoyu Zhang 1, Qihuan Li 1, Haifeng Hou 2, Dong Li 2, Wei Wang 1,2,3, Youxin Wang 1
1Beijing Key Laboratory of Clinical Epidemiology, School of Public Health, Capital Medical University, 2School of Public Health, Shandong First Medical University & Shandong Academy of Medical Sciences, 3School of Medical and Health Sciences, Edith Cowan University

Immunoglobulin G (IgG) N-glycan is characterized using hydrophilic interaction chromatography UPLC. In addition, the structure of IgG N-glycan is clearly separated. Presented here is an introduction to this experimental method so that it can be widely used in research settings.

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Neuroscience

Cochlear Surface Preparation in the Adult Mouse
Qiao-Jun Fang 1,2, Fan Wu 1, Renjie Chai 2, Su-Hua Sha 1
1Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 2MOE Key Laboratory of Developmental Genes and Human Disease, Institute of Life Sciences, Southeast University

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

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Neuroscience

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing
Lu Zhou 1, Qingyun Li 1
1Department of Neurobiology, Stanford University School of Medicine

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

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JoVE Core

A Reversible Silicon Oil-Induced Ocular Hypertension Model in Mice
Jie Zhang 1,2, Fang Fang 1,3, Liang Li 1, Haoliang Huang 1, Hannah C. Webber 1, Yang Sun 1,4, Vinit B. Mahajan 1,4, Yang Hu 1
1Department of Ophthalmology, Stanford University School of Medicine, 2Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 3Department of Ophthalmology, Second Xiangya Hospital of Central South University, 4Department of Ophthalmology, Veterans Affairs Palo Alto Health Care

Here, we present a protocol to induce ocular hypertension and glaucomatous neurodegeneration in mouse eyes by intracameral injection of silicone oil and the procedure for silicone oil removal from the anterior chamber to return elevated intraocular pressure to normal.

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Bioengineering

Design of a Biocompatible Drug-Eluting Tracheal Stent in Mice with Laryngotracheal Stenosis
Madhavi Duvvuri 1, Kevin Motz 2, Hsiu-Wen Tsai 2, Ioan Lina 2, Dacheng Ding 2, Andrew Lee 2, Alexander T. Hillel 2
1Department of General Surgery, University of California, San Francisco, 2Department of Otolaryngology Head and Neck Surgery, Johns Hopkins School of Medicine

Laryngotracheal stenosis results from pathologic scar deposition that critically narrows the tracheal airway and lacks effective medical therapies. Using a PLLA-PCL (70% poly-L-lactide and 30% polycaprolactone) stent as a local drug delivery system, potential therapies aimed at decreasing scar proliferation in the trachea can be studied.

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Genetics

Combining Laser Capture Microdissection and Microfluidic qPCR to Analyze Transcriptional Profiles of Single Cells: A Systems Biology Approach to Opioid Dependence
Sean J. O'Sullivan 1,2, Beverly A.S. Reyes 3, Rajanikanth Vadigepalli 1, Elisabeth J. Van Bockstaele 3, James S. Schwaber 1
1Daniel Baugh Institute for Functional Genomics and Computational Biology, Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, 2Sidney Kimmel Medical College, Thomas Jefferson University, 3Department of Pharmacology & Physiology, Drexel University College of Medicine

This protocol explains how to collect single neurons, microglia, and astrocytes from the central nucleus of the amygdala with high accuracy and anatomic specificity using laser capture microdissection. Additionally, we explain our use of microfluidic RT-qPCR to measure a subset of the transcriptome of these cells.

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Developmental Biology

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells
Wei Feng 1, Andrew Przysinda 1, Guang Li 1
1Department of Developmental Biology, University of Pittsburgh School of Medicine

Here we presented a multiplexed single cell mRNA sequencing method to profile gene expression in mouse embryonic tissues. The droplet-based single cell mRNA sequencing (scRNA-Seq) method in combination with multiplexing strategies can profile single cells from multiple samples simultaneously, which significantly reduces reagent costs and minimizes experimental batch effects.

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Developmental Biology

Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils
Soah Lee *1,2,3, Huaxiao Yang *1,2,3, Caressa Chen *1,2,3, Sneha Venkatraman 1,2,3, Adrija Darsha 1,2,3, Sean M. Wu 1,2,3, Joseph C. Wu 1,2,3, Timon Seeger 1,2,3,4,5
1Stanford Cardiovascular Institute, Stanford University School of Medicine, 2Department of Medicine, Division of Cardiovascular Medicine, Stanford University, 3Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, 4Department of Medicine III, University Hospital Heidelberg, 5German Centre for Cardiovascular Research (DZHK), Partner Site Heidelberg/Mannheim

This protocol introduces a lithography-free micropatterning method that is simple and accessible to those with a limited bioengineering background. This method utilizes customized laser-cut stencils to micropattern extracellular matrix proteins in a shape of interest for modulating cell morphologies. The procedure for micropatterning is demonstrated using induced pluripotent stem cell derived cardiomyocytes.

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Medicine

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model
Michaela Olthoff 1, Federico Franchi 1, Karen M. Peterson 1, Ramasamy Paulmurugan 2, Martin Rodriguez-Porcel 1
1Department of Cardiovascular Medicine, Mayo Clinic, 2Department of Radiology and Molecular Imaging Program at Stanford (MIPS), Stanford University School of Medicine

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

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Neuroscience

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions
Benedetta Assetta *1, Changyong Tang *1,2, Jing Bian *3, Ryan O'Rourke 1, Kevin Connolly 1, Thomas Brickler 3, Sundari Chetty 3,4, Yu-Wen Alvin Huang 1,5,6
1Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, 2Department of Neurology, The Third Affiliated Hospital of Sun Yat-Sen University, 3Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, 4Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, 5Department of Neurology, Warren Alpert Medical School of Brown University, 6Center for Translational Neuroscience, Robert J. and Nancy D. Carney Institute for Brain Science and Brown Institute for Translational Science, Brown University

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer’s disease.

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Bioengineering

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells
Dilip Thomas 1, Hyeonyu Kim 1, Nicole Lopez 1, Joseph C. Wu 1,2,3
1Stanford Cardiovascular Institute, Stanford University School of Medicine, 2Department of Medicine, Division of Cardiovascular Medicine, Stanford University School of Medicine, 3Department of Radiology, Stanford University School of Medicine

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

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Immunology and Infection

Profiling of the Human Natural Killer Cell Receptor-Ligand Repertoire
Elena Vendrame *1, Julia L. McKechnie *1,2, Thanmayi Ranganath 1, Nancy Q. Zhao 1,2, Arjun Rustagi 1, Rosemary Vergara 1, Geoffrey T. Ivison 1,2, Lisa M. Kronstad 1, Laura J. Simpson 1, Catherine A. Blish 1,2,3
1Department of Medicine, Stanford University School of Medicine, 2Program in Immunology, Stanford University School of Medicine, 3Chan-Zuckerberg BioHub

Here we design two complementary mass cytometry (CyTOF) panels and optimize a CyTOF staining protocol with the aim of profiling the natural killer cell receptor and ligand repertoire in the setting of viral infections.

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Biology

Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization
Xianrui Cheng 1,2, James E. Ferrell, Jr. 1,3
1Department of Chemical and Systems Biology, Stanford University School of Medicine, 2Current Address: Department of Biological Sciences, University of Southern California, 3Department of Biochemistry, Stanford University School of Medicine

We describe a method for the preparation and live imaging of undiluted cytoplasmic extracts from Xenopus laevis eggs.

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Biology

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission
György Vámosi 1, Sarah Miller 2, Molika Sinha 2, Maria Kristha Fernandez 2, Gabor Mocsár 1, Malte Renz 2
1Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, 2Gynecologic Oncology Division, Stanford University School of Medicine

Förster Resonance Energy Transfer (FRET) between two fluorophore molecules can be used for studying protein interactions in the living cell. Here, a protocol is provided as to how to measure FRET in live cells by detecting sensitized emission of the acceptor and quenching of the donor molecule using confocal laser scanning microscopy.

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Cancer Research

Sentinel Lymph Node Mapping and Biopsy for Endometrial Cancer at Early Stage with Laparoscopy
Bo Wang 1, Yu Xue 1, Qian Wang 1, Yan Xu 1, Xiaojun Chen 1, Chao Wang 1
1Obstetrics and Gynecology Hospital of Fudan University

This protocol describes the identification and resection of sentinel lymph nodes to make the operation as easy and minimally invasive as possible.

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Developmental Biology

Applications of RNA Interference in American Cockroach
Liang Li *1, Andi Jing *1, Minxin Xie 1, Sheng Li 1,2, Chonghua Ren 1,2
1Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Institute of Insect Science and Technology & School of Life Sciences, South China Normal University, 2Guangmeiyuan R&D Center, Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, South China Normal University

The present protocol describes step-by-step guidelines for the RNAi operation techniques in P. americana.

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Biology

Structure-Based Simulation and Sampling of Transcription Factor Protein Movements along DNA from Atomic-Scale Stepping to Coarse-Grained Diffusion
Chao E *1, Liqiang Dai *1,2, Jiaqi Tian 3,4, Lin-Tai Da 4, Jin Yu 5,6,7
1Beijing Computational Science Research Center, 2Shenzhen JL Computational Science and Applied Research Institute, 3School of Medical Informatics and Engineering, Xuzhou Medical University, 4Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, 5Department of Physics and Astronomy, University of California, Irvine, 6Department of Chemistry, University of California, Irvine, 7NSF-Simons Center for Multiscale Cell Fate Research, University of California, Irvine

The goal of this protocol is to reveal structural dynamics of one-dimensional diffusion of protein along DNA, using a plant transcription factor WRKY domain protein as an exemplary system. To do this, both atomistic and coarse-grained molecular dynamics simulations along with extensive computational samplings have been implemented.

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Neuroscience

Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice
Caitlyn M. Gaffney 1, Gabriella Muwanga 2, Huaishuang Shen 2, Vivianne L. Tawfik 2, Andrew J. Shepherd 2
1Laboratories of Neuroimmunology, Department of Symptom Research, and the MD Anderson Pain Research Consortium, University of Texas MD Anderson Cancer Center, 2Department of Anesthesiology, Perioperative and Pain Medicine, Stanford University School of Medicine

The mechanical conflict-avoidance assay is used as a non-reflexive readout of pain sensitivity in mice which can be used to better understand affective-motivational responses in a variety of mouse pain models.

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Biochemistry

Exploring Biomolecular Interaction Between the Molecular Chaperone Hsp90 and Its Client Protein Kinase Cdc37 using Field-Effect Biosensing Technology
Yana Lerner *1, Surya Sukumaran *1, Mei-Sze Chua 2, Samuel K. So 2, Nir Qvit 1
1The Azrieli Faculty of Medicine in the Galilee, Bar-Ilan University, 2Asian Liver Center, Department of Surgery, Stanford University School of Medicine

Field-effect biosensing (FEB) is a label-free technique for detecting biomolecular interactions. It measures the electric current through the graphene biosensor to which the binding targets are immobilized. The FEB technology was used to evaluate biomolecular interactions between Hsp90 and Cdc37 and a strong interaction between the two proteins was detected.

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Neuroscience

The Tibial Fracture-Pin Model: A Clinically Relevant Mouse Model of Orthopedic Injury
Gabriella P. B. Muwanga 1,2, Janelle Siliezar-Doyle 1,2, Angel Amadeus Ortiz 1, Jasmine Kaslow 1, Elena S. Haight 1, Vivianne L. Tawfik 1
1Department of Anesthesiology, Perioperative and Pain Medicine, Stanford University School of Medicine, 2Neurosciences Graduate Program, Stanford University School of Medicine

The tibial fracture-pin model is a clinically relevant model of orthopedic trauma comprising a unilateral open tibial fracture with intramedullary nail internal fixation and simultaneous injury to the tibialis anterior muscle. Thermal sensitivity in this model can be measured using a 45 s hot plate paradigm.

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Neuroscience

Investigating Drivers of Antireward in Addiction Behavior with Anatomically Specific Single-Cell Gene Expression Methods
Sean J. O'Sullivan *1,2,3, Ankita Srivastava *1, Rajanikanth Vadigepalli 1, James S. Schwaber 1
1Daniel Baugh Institute for Functional Genomics and Computational Biology, Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, 2Sidney Kimmel Medical College, Thomas Jefferson University, 3Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine

The combination of laser capture microdissection and microfluidic RT-qPCR provides anatomic and biotechnical specificity in measuring the transcriptome in single neurons and glia. Applying creative methods with a system's biology approach to psychiatric disease may lead to breakthroughs in understanding and treatment such as the neuroinflammation antireward hypothesis in addiction.

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Biology

Protocols for CRISPR/Cas9 Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis
Jinxi Yuan 1, Jie Zhang 1, Yan Zhang 1, WuYun QiQiGe 1, Wei Liu 2, Shanchun Yan 1, Guirong Wang 2
1Key Laboratory of Sustainable Forest Ecosystem Management - Ministry of Education, Northeast Forestry University, 2Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences

This paper presents the step-by-step protocols for CRISPR/Cas9 mutagenesis of the Oriental fruit fly Bactrocera dorsalis. Detailed steps provided by this standardized protocol will serve as a useful guide for generating mutant flies for functional gene studies in B. dorsalis.

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Medicine

Technical Applications of Microelectrode Array and Patch Clamp Recordings on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes
Shane Rui Zhao 1,2, Gema Mondéjar-Parreño 1,2, Dong Li 1,2, Mengcheng Shen 1,2, Joseph C. Wu 1,2,3
1Stanford Cardiovascular Institute, Stanford University School of Medicine, 2Division of Cardiovascular Medicine, Department of Medicine, Stanford University School of Medicine, 3Department of Radiology, Stanford University School of Medicine

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a promising in vitro model for drug-induced cardiotoxicity screening and disease modeling. Here, we detail a protocol for measuring the contractility and electrophysiology of hiPSC-CMs.

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Medicine

Chronic Ovine Model of Right Ventricular Failure and Functional Tricuspid Regurgitation
Boguslaw Gaweda 1,2, Artur Iwasieczko 1,2, Manikantam Gaddam 3,4, Jared D. Bush 3, Brian MacDougal 3, Tomasz A. Timek 1
1Division of Cardiothoracic Surgery, Spectrum Health, 2Clinical Department of Cardiac Surgery, Clinical District Hospital no 2, Faculty of Medicine, University of Rzeszow, 3Research Department, Meijer Heart and Vascular Institute at Spectrum Health, 4Department of Urology, Stanford University School of Medicine

Right ventricular failure and functional tricuspid regurgitation are associated with left-sided heart disease and pulmonary hypertension, which contribute significantly to morbidity and mortality in patients. Establishing a chronic ovine model to study right ventricular failure and functional tricuspid regurgitation will help in understanding their mechanisms, progression, and possible treatments.

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Cancer Research

Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells
Tommaso Sconocchia 1, Johannes Foßelteder 1, Thomas Köhnke 2, Ravindra Majeti 2, Andreas Reinisch 1,3
1Division of Hematology, Department of Internal Medicine, Medical University of Graz, 2Division of Hematology, Stanford Cancer Institute, Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, 3Department of Blood Group Serology and Transfusion Medicine, Medical University of Graz

Novel strategies to faithfully model somatic mutations in hematopoietic stem and progenitor cells (HSPCs) are necessary to better study hematopoietic stem cell biology and hematological malignancies. Here, a protocol to model heterozygous gain-of-function mutations in HSPCs by combining the use of CRISPR/Cas9 and dual rAAV donor transduction is described.

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Bioengineering

High-Performance Graphene-Modified Sensing Chip for SARS-CoV-2 Detection
Parshant Kumar Sharma 1,2,3, Ebrahim Mostafavi 4,5, Nam-Young Kim 1,2,3, Thomas J. Webster 6, Ajeet Kaushik 7,8
1RFIC Bio Centre, Kwangwoon University, 2Department of Electronics Engineering, Kwangwoon University, 3NDAC Centre, Kwangwoon University, 4Stanford Cardiovascular Institute, Stanford University School of Medicine, 5Department of Medicine, Stanford University School of Medicine, 6Department of Biomedical Engineering, Hebei University of Technology, 7NanoBioTech Laboratory, Department of Environmental Engineering, Florida Polytechnic University, 8School of Engineering, University of Petroleum and Energy Studies (UPES)

The present protocol describes the fabrication of low-cost biosensing prototypes based on useful nanosystems for accurately detecting viral proteins (at the Fg level). Such a tiny sensor platform allows for point-of-care applications that can be integrated with the Internet of Medical Things (IoMT) to meet telemedicine objectives.

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Medicine

Corneal and Limbal Alkali Injury Induction Using a Punch-Trephine Technique in a Mouse Model
Athar Shadmani 1, Hala Shakib Dhowre 1, Ozlem Ercal 1, Xiang Qi Meng 2, Albert Y. Wu 1
1Department of Ophthalmology, Stanford University School of Medicine, 2McGill University Faculty of Medicine and Health Sciences

This protocol describes a method to induce an accurate and reproducible corneal and limbal alkali injury in a mouse model. The protocol is advantageous as it allows for an evenly distributed injury to the highly curved mouse cornea and limbus.

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Medicine

Simplified Intrafemoral Injections Using Live Mice Allow for Continuous Bone Marrow Analysis
Yusuke Nakauchi 1, Asiri Ediriwickrema 1, Daniel Martinez-Krams 1, Feifei Zhao 1, Athreya Rangavajhula 1, Daiki Karigane 1, Ravindra Majeti 1
1Department of Medicine, Division of Hematology, Cancer Institute, and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine

This protocol describes the intrafemoral injection of a few hematopoietic or leukemic stem cells, including gene-edited cells, in murine xenograft models, which will not only enable the quick and safe transplantation of cells but also serial analyses of the bone marrow.

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Cancer Research

Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model
Kerem Nernekli *1, Dilyana B. Mangarova *1, Yifeng Shi 2, Zahra Shokri Varniab 1, Edwin Chang 1, Oguz Ziya Tikenogullari 3, Laura Pisani 1, Grigory Tikhomirov 2, Gordon Wang 4, Heike E. Daldrup-Link 1
1Molecular Imaging Program at Stanford (MIPS), Department of Radiology, Stanford University School of Medicine, 2Department of Electrical Engineering and Computer Sciences, University of California, Berkeley, 3Department of Mechanical Engineering, Stanford University, 4Department of Psychiatry and Behavioral Sciences, Stanford University, Wu Tsai Neuroscience Institute, Stanford University

We present a novel approach for two-photon microscopy of the tumor delivery of fluorescent-labeled iron oxide nanoparticles to glioblastoma in a mouse model.

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