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78 ARTICLES PUBLISHED IN JoVE

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Biology

Ex vivo Mechanical Loading of Tendon
Krishna Asundi 1, David Rempel 2
1Department of Bioengineering, University of California, Berkeley , 2Department of Bioengineering, University of California, Berkeley; Division of Occupational Medicine, University of California, San Francisco

A new in vitro system for simultaneously loading four tendons in culture is described.

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Biology

Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice
Melanie P. Matheu 1, Ian Parker 2, Michael D. Cahalan 1
1Department of Physiology and Biophysics, University of California, Irvine (UCI), 2Department of Neurobiology and Behaviour, University of California, Irvine (UCI)

Two-photon imaging has uncovered lymphocyte motility and cellular interactions within the lymph node under basal conditions and durring an immune response 1. Here, we demonstrate adoptive transfer of T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node.

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Biology

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification
Melanie P. Matheu 1, Michael D. Cahalan 1
1Department of Physiology and Biophysics, University of California, Irvine (UCI)

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically ≥ 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

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Biology

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging
Melanie P. Matheu 1, Debasish Sen 1, Michael D Cahalan 1, Ian Parker 2
1Department of Physiology and Biophysics, University of California, Irvine (UCI), 2Department of Neurobiology and Behaviour, University of California, Irvine (UCI)

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

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Biology

Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH
Melanie P. Matheu 1, Christine Beeton 1, Ian Parker 2, K. George Chandy 1, Michael D. Cahalan 1
1Department of Physiology and Biophysics, University of California, Irvine (UCI), 2Department of Neurobiology and Behavior, University of California, Irvine (UCI)

Here we demonstrate a method for inducing and recording the progress of a delayed type-hypersensitivity (DTH) reaction in the rat ear. This is followed by a demonstration of the preparation of rat ear tissue for two-photon imaging of the effector / memory T cell response.

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Biology

Murine Model of Hindlimb Ischemia
Hiroshi Niiyama 1, Ngan F. Huang 1, Mark D. Rollins 2, John P. Cooke 1
1Division of Cardiovascular Medicine, Stanford University , 2Department of Anesthesiology, University of California, San Francisco

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

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Neuroscience

A Novel Method for Assessing Proximal and Distal Forelimb Function in the Rat: the Irvine, Beatties and Bresnahan (IBB) Forelimb Scale
Karen-Amanda Irvine 1, Adam R. Ferguson 1, Kathleen D. Mitchell 1, Stephanie B. Beattie 1, Michael S. Beattie 1, Jacqueline C. Bresnahan 1
1Department of Neurological Surgery, University of California, San Francisco

Here we will describe a rodent behavioral assay that can detect recovery of both proximal and distal forelimb function including digit movements during a naturally occurring behavior that does not require extensive training or deprivation to enhance motivation.

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Immunology and Infection

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens
Eunice C. Chen 1, Steve A. Miller 1, Joseph L. DeRisi 1,2, Charles Y. Chiu 1,2
1Department of Laboratory Medicine, University of California, San Francisco, 2Division of Infectious Diseases, University of California, San Francisco

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

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Neuroscience

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain
Zhiqiang Dong 1, Mahendra Wagle 1, Su Guo 1
1Department of Bioengineering and Therapeutic Sciences, Programs in Human Genetics and Biological Sciences , University of California San Francisco

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

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Medicine

Guide Wire Assisted Catheterization and Colored Dye Injection for Vascular Mapping of Monochorionic Twin Placentas
Eric B. Jelin 1, Samuel C. Schecter 1, Kelly D. Gonzales 1, Shinjiro Hirose 1, Hanmin Lee 1, Geoffrey A. Machin 2, Larry Rand 3, Vickie A. Feldstein 4
1Division of Pediatric and Fetal Surgery, Department of Surgery, University of California, San Francisco, 2Department of Pathology, University of Alberta, 3Department of Obstretics and Gynecology, University of California, San Francisco, 4Department of Radiology, University of California, San Francisco

Vascular mapping of monochorionic (MC) twin placentas after birth provides a means for detailed demonstration of vascular connections between the twins’ circulations. Imbalance of these connections is thought to play a pivotal role in the development of complications of MC twinning including twin-to-twin transfusion syndrome.

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Neuroscience

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis
Espen J. Walker 1, Fanxia Shen 1, William L. Young 1,2,3, Hua Su 1
1Center for Cerebrovascular Research, Department of Anesthesia and Perioperative Care, University of California, San Francisco, 2Department of Neurological Surgery, University of California, San Francisco, 3Department of Neurology, University of California, San Francisco

In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse brain.

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Biology

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes
Yanning Wu 1, Shuo Wang 1, Chunying Li 1,2,3
1Department of Biochemistry & Molecular Biology, Wayne State University School of Medicine, 2Cardiovascular Research Institute, Wayne State University School of Medicine, 3Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine

Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.

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Medicine

Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis.
Shun-Tai Yang 1, Ana Rodriguez-Hernandez 2, Espen J. Walker 1, William L. Young 1,2,3, Hua Su 1, Michael T. Lawton 2
1Department of Anesthesia and Perioperative Care and Center for Cerebrovascular Research, University of California, San Francisco, 2Department of Neurological Surgery, University of California, San Francisco, 3Department of Neurology, University of California, San Francisco

We describe a method for creating a reliable model of cerebral venous hypertension in the adult mouse. This model has been widely described and tested in the rat. This new counterpart in the mice opens the possibility of using genetic modified animals and thereby broadens the applications of the model.

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Bioengineering

Insertion of Flexible Neural Probes Using Rigid Stiffeners Attached with Biodissolvable Adhesive
Sarah H. Felix 1, Kedar G. Shah 1, Vanessa M. Tolosa 1, Heeral J. Sheth 1, Angela C. Tooker 1, Terri L. Delima 1, Shantanu P. Jadhav 2, Loren M. Frank 2, Satinderpall S. Pannu 1
1Materials Engineering Division, Lawrence Livermore National Laboratory, 2UCSF Center for Integrative Neuroscience and the Department of Physiology, University of California, San Francisco

Insertion of flexible neural microelectrode probes is enabled by attaching probes to rigid stiffeners with polyethylene glycol (PEG). A unique assembly process ensures uniform and repeatable attachment. After insertion into tissue, the PEG dissolves and the stiffener is extracted. An in vitro test method evaluates the technique in agarose gel.

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Immunology and Infection

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions
Kevin Wilhelmsen 1, Katherine Farrar 1,2, Judith Hellman 1
1Department of Anesthesia and Perioperative Care, University of California, San Francisco, 2Graduate Program in Biomedical Sciences, University of California, San Francisco

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

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Biology

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists
Hao Li *1, Wei Dou *2, Emil Padikkala 1, Sridhar Mani 1
1Department of Genetics, Albert Einstein College of Medicine , 2Shanghai Key Laboratory of Complex Prescription and MOE Key Laboratory for Standardization of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

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Biology

The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis
Jillian M. Silva 1, Martin McMahon 1
1Diller Cancer Research Building, University of California, San Francisco

This protocol explores the latest advancements in performing Western blot analyses. These novel modifications employ a Bis-Tris gel system with a 35 min electrophoresis run time, a 7 min dry blotting transfer system, and infrared fluorescent protein detection and imaging that generates higher resolution, quality, sensitivity, and improved accuracy of Western data.

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Biology

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor
Miquella G. Chavez 1,2, Jimmy Hu 1, Kerstin Seidel 1, Chunying Li 1,3, Andrew Jheon 1, Adrien Naveau 4,5,6, Orapin Horst 1,7, Ophir D. Klein 1,8
1Department of Orofacial Sciences and Program in Craniofacial and Mesenchymal Biology, University of California, San Francisco, 2Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, 3Department of Pathology and Research Center, Zhongshan Hospital of Dalian University, 4Université Paris Descartes, Sorbonne Paris Cite, UMR S872, 5Centre de Recherche des Cordeliers, Université Pierre et Marie Curie, UMR S872, 6INSERM U872, 7Division of Endodontics, Department of Preventive and Restorative Dental Sciences, University of California, San Francisco, 8Department of Pediatrics and Institute for Human Genetics, University of California, San Francisco

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

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Medicine

RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma
Hongwei Wang 1, Mindy Xiao-Ming Wang 1, Nan Su 1, Li-chong Wang 1, Xingyong Wu 1, Son Bui 1, Allissa Nielsen 1, Hong-Thuy Vo 1, Nina Nguyen 1, Yuling Luo 1, Xiao-Jun Ma 1
1Advanced Cell Diagnostics, Inc.

The presence of high-risk HPV in head and neck tumor tissue is associated with favorable outcomes. The recently developed RNA in situ hybridization technique called RNAscope allows direct visualization of HPV E6/E7 mRNA in FFPE tissue sections.

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Bioengineering

Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding
Philip Chung 1, J. Alex Heller 1, Mozziyar Etemadi 1, Paige E. Ottoson 2, Jonathan A. Liu 3, Larry Rand 2, Shuvo Roy 1
1Department of Bioengineering & Therapeutic Sciences, University of California, San Francisco, 2Department of Obstetrics, Gynecology & Reproductive Sciences, University of California, San Francisco, 3Keck School of Medicine, University of Southern California

We have devised a method for low-cost and rapid prototyping of liquid elastomer rubber injection molded devices by using fused deposition modeling 3D printers for mold design and a modified desiccator as a liquid injection system.

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JoVE Core

Ultrasound Assessment of Endothelial-Dependent Flow-Mediated Vasodilation of the Brachial Artery in Clinical Research
Hugh Alley 1,2,3, Christopher D. Owens 1,2,3, Warren J. Gasper 1,2,3, S. Marlene Grenon 1,2,3
1Department of Surgery, University of California, San Francisco, 2Department of Surgery, Veterans Affairs Medical Center, San Francisco, 3VipeRx Lab, Veterans Affairs Medical Center, San Francisco

Endothelial dysfunction is associated with numerous disease states and is predictive of adverse cardiovascular events in humans. Flow-mediated vasodilation (FMD) is a non-invasive ultrasound method of evaluating endothelial function. Methodological choices and operator experience may affect results. A systematic approach to FMD in human studies is discussed here.

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Neuroscience

Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane
Omar Akil 1, Stephanie L. Rouse 1, Dylan K. Chan 1, Lawrence R. Lustig 1
1Department of Otolaryngology-Head and Neck Surgery, University of California, San Francisco

The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane.

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Bioengineering

Production and Targeting of Monovalent Quantum Dots
Daeha Seo *1,2,3, Justin Farlow *4,5,6, Kade Southard 1,4,7, Young-wook Jun 1,7, Zev J. Gartner 4,5,6,7
1Department of Otolaryngology, University of California, San Francisco, 2Department of Chemistry, University of California, Berkeley, 3Materials Science Division, Lawrence Berkeley National Laboratory, 4Department of Pharmaceutical Chemistry, University of California, San Francisco, 5Tetrad Graduate Program, University of California, San Francisco, 6Center for Systems and Synthetic Biology, University of California, San Francisco, 7Chemistry and Chemical Biology Graduate Program, University of California, San Francisco

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

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Behavior

A Cognitive Paradigm to Investigate Interference in Working Memory by Distractions and Interruptions
Jacki Janowich 1, Jyoti Mishra 2, Adam Gazzaley 2,3,4
1Department of Psychology, University of New Mexico, 2Department of Neurology, University of California, San Francisco, 3Department of Physiology, Center for Integrative Neuroscience, University of California, San Francisco, 4Department of Psychiatry, University of California, San Francisco

A novel cognitive paradigm is developed to elucidate behavioral and neural correlates of interference by to-be-ignored distractors versus interference by to-be-attended interruptors during a working memory task. In this manuscript, several variants of this paradigm are detailed, and data obtained with this paradigm in younger/older adult participants is reviewed.

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Developmental Biology

Mechanical Vessel Injury in Zebrafish Embryos
Hilary Clay 1, Shaun R. Coughlin 1
1Cardiovascular Research Institute, University of California

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

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Biology

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry
Heather Inglis 1, Philip Norris 1,2,3, Ali Danesh 1,3
1Blood Systems Research Institute, 2Department of Medicine, University of California, San Francisco, 3Department of Laboratory Medicine, University of California, San Francisco

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

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Neuroscience

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord
Jason G. Weinger 1, Milton L. Greenberg 2, Melanie P. Matheu 4, Ian Parker 3, Craig M. Walsh 1, Thomas E. Lane 5, Michael D. Cahalan 2
1Molecular Biology and Biochemistry, University of California, Irvine, 2Physiology and Biophysics, University of California, Irvine, 3Neurobiology and Behavior, University of California, Irvine, 4University of California San Francisco Diabetes Center, University of California, San Francisco, 5Pathology, University of Utah

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

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Developmental Biology

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart
Lisa D. Wilsbacher 1,2, Shaun R. Coughlin 2
1Feinberg Cardiovascular Research Institute, Northwestern University, 2Cardiovascular Research Institute, University of California, San Francisco

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

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Medicine

Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma
Aaron J. Clark 1, Shayan Fakurnejad 2, Quanhong Ma 2, Rintaro Hashizume 2,3
1Department of Neurological Surgery, University of California San Francisco, 2Department of Neurological Surgery, Northwestern University Feinberg School of Medicine, 3Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine

GL261 glioma cells provide a useful immunocompetent animal model of glioblastoma. The goals of this protocol are to demonstrate proper techniques for monitoring intracranial tumor growth using in vivo bioluminescence imaging, and to verify the utility of luciferase-modified GL261 cells for studying tumor immunology and immunotherapeutic approaches for treating glioblastoma.

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Bioengineering

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device
Russell H. Cole 1, Tuan M. Tran 2, Adam R. Abate 3,4
1Department of Pharmaceutical Chemistry, University of California, San Francisco, 2Joint UCSF/UCB Bioengineering Graduate Group, University of California, San Francisco, 3Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, 4California Institute for Quantitative Biosciences, University of California, San Francisco

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 µm.

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Education

Measuring Cardiac Autonomic Nervous System (ANS) Activity in Toddlers - Resting and Developmental Challenges
Nicole R. Bush 1,2, Zoe K. Caron 1, Katherine S. Blackburn 1, Abbey Alkon 3
1Department of Psychiatry, University of California, San Francisco, 2Department of Pediatrics, University of California, San Francisco, 3School of Nursing, University of California, San Francisco

We describe the methods for continuous monitoring of the autonomic nervous system under resting and challenge conditions with 18 month old children. Results revealed that this protocol can produce meaningful physiological responses in both branches of the autonomic nervous system and elicit significant individual variability in patterns of responses.

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Neuroscience

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons
Ji-Yong Kang 1, Daichi Kawaguchi 2, Lei Wang 3
1Department of Neuroscience, School of Medicine, Tufts University, 2Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, 3Department of Pharmaceutical Chemistry and the Cardiovascular Research Institute, University of California, San Francisco

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

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Bioengineering

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers
Russell H. Cole 1, Zev J. Gartner 1, Adam R. Abate 2
1Department of Pharmaceutical Chemistry, University of California, San Francisco, 2Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biosciences, University of California, San Francisco

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

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Biology

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes
Justin Judd 1, Jonathan Lovas 1, Guo N. Huang 1
1Cardiovascular Research Institute, University of California, San Francisco

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

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Immunology and Infection

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface
Gabrielle A. Rizzuto 1,2, Mirhan Kapidzic 3, Matthew Gormley 3, Anna I. Bakardjiev 1,4,5
1Benioff Children’s Hospital, 2Department of Pathology, University of California, San Francisco, 3Center for Reproductive Sciences and Department of Obstetrics, University of California, San Francisco, 4Program in Microbial Pathogenesis and Host Defense, University of California, San Francisco, 5Biomedical Sciences Program, University of California, San Francisco

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

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JoVE Journal

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood
Clovis S. Palmer 1,2,3, Joshua J. Anzinger 4, Tiffany R. Butterfield 4, Joseph M. McCune 5, Suzanne M. Crowe 1,2,6
1Centre for Biomedical Research, Macfarlane Burnet Institute for Medical Research and Public Health, 2Department of Infectious Diseases, Monash University, 3Department of Microbiology and Immunology, University of Melbourne, 4Department of Microbiology, The University of the West Indies, 5Division of Experimental Medicine, Department of Medicine, University of California, San Francisco, 6Department of Medicine, Monash University

Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.

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Biochemistry

Patch Clamp Recordings on Intact Dorsal Root Ganglia from Adult Rats
Kerui Gong 1, Peter T. Ohara 2, Luc Jasmin 1
1Department of Oral and Maxillofacial Surgery, University of California, San Francisco, 2Department of Anatomy, University of California, San Francisco

This manuscript describes how to prepare intact dorsal root ganglia for patch clamp recordings. This preparation maintains the microenvironment for neurons and satellite glial cells, thus avoiding the phenotypic and functional changes seen using dissociated DRG neurons.

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Biochemistry

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram
Jonathan A. Coleman 1, Evan M. Green 2, Eric Gouaux 1,3
1Vollum Institute, Oregon Health & Science University, 2Graduate Group in Biophysics, University of California, San Francisco, 3Howard Hughes Medical Institute, Oregon Health & Science University

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

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Medicine

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats
Amara Larpthaveesarp 1, Fernando F. Gonzalez 1
1Department of Pediatrics, University of California, San Francisco

Neonatal stroke is a significant cause of early brain injury requiring a translational model with consistent focal injury patterns and high reproducibility in order to enable study. This study describes the detailed surgical procedure for creating a non-hemorrhagic, unilateral focal ischemia-reperfusion injury in full-term-equivalent rodents.

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Developmental Biology

A Modified Trier Social Stress Test for Vulnerable Mexican American Adolescents
Megan M. Johnson 1, Julianna Deardorff 1, Kimberly Parra 1, Abbey Alkon 2, Brenda Eskenazi 1, Elizabeth Shirtcliff 3
1Center for Environmental Research and Children's Health (CERCH), Berkley School of Public Health, University of California, Berkeley, 2San Francisco (UCSF) School of Nursing, University of California, San Francisco, 3Human Development and Family Studies, Iowa State University

Here, we present a protocol that provoked cortisol reactivity in a vulnerable adolescent Mexican American sample utilizing a modified version of the Trier Social Stress Test (TSST). Saliva samples were collected at baseline, 15, 30, and 45 min post-TSST onset. Future research could utilize this modified TSST with vulnerable youth.

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Biology

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells
Ke Zou 1,2, Diana S. Ren 2, Qi Ou-yang 1,3, Hao Li 2, Jiashun Zheng 2
1The State Key Laboratory for Artificial Microstructures and Mesoscopic Physics, School of Physics, Peking University, 2Department of Biochemistry and Biophysics, University of California, San Francisco, 3Peking-Tsinghua Center for Life Sciences at Center for Quantitative Biology, Academy for Advanced Interdisciplinary Studies, Peking University

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

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Immunology and Infection

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation
Misty M. Montoya 1, K. Mark Ansel 1
1Department of Microbiology & Immunology, Sandler Asthma Basic Research Center, University of California, San Francisco

Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

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Environment

Data Processing Methods for 3D Seismic Imaging of Subsurface Volcanoes: Applications to the Tarim Flood Basalt
Lei Wang 1, Wei Tian 2, Yongmin Shi 2
1College of Engineering, Peking University, 2School of Earth and Space Sciences, Peking University

Three-dimensional (3D) reflection seismology is a powerful method for imaging subsurface volcanoes. By using industrial 3D seismological data from the Tarim Basin, we illustrate how to extract the sills and the conduits of subsurface volcanoes from seismic data cubes.

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JoVE Core

A Novel Method for Involving Women of Color at High Risk for Preterm Birth in Research Priority Setting
Linda S. Franck 1,2, Monica R. McLemore 1,2, Norlissa Cooper 1, Baylee De Castro 3, Anastasia Y. Gordon 4, Schyneida Williams 5, Shanell Williams 2, Larry Rand 2,3
1School of Nursing, University of California, San Francisco, 2UCSF California Preterm Birth Initiative, University of California, San Francisco, 3School of Medicine, University of California, San Francisco, 4San Francisco Black Infant Health Program, 5Homeless Prenatal Program, San Francisco, CA

This manuscript describes the Research Prioritization by Affected Communities (RPAC) protocol and findings from its use with women at risk for preterm birth. Using the protocol, women identified and prioritized their unanswered questions about pregnancy, birth and neonatal care aimed at influencing research priority setting by funders and researchers.

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Developmental Biology

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue
Sumitra Tatapudy 1, Marimar Benitez 1, Todd Nystul 1
1Departments of Anatomy and OB/GYN-RS, University of California, San Francisco

We provide a protocol for imaging intracellular pH of an epithelial stem cell lineage in live Drosophila ovarian tissue. We describe methods to generate transgenic flies expressing a pH biosensor, mCherry::pHluorin, image the biosensor using quantitative fluorescence imaging, generate standard curves, and convert fluorescence intensity values to pH values.

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JoVE Journal

Differential Effects of Lipid-lowering Drugs in Modulating Morphology of Cholesterol Particles
Shanmugavel Madasamy 1, David Liu 1, Jason Lundry 1, Benjamin Alderete 2, Raymond Kong 2, J. Paul Robinson 3, Alan H.B. Wu 1,4, Edward P. Amento 5
1Plaxgen Inc, 2Millipore Sigma, 3Cytometry Laboratories, Purdue University, 4San Francisco General Hospital, 2M16 Clinical Chemistry, University of California, San Francisco, 5Molecular Medicine Research Institute

The objective of this study was to evaluate in vitro lipid-lowering drug effects in modulating the morphology of cholesterol particles. Comparison of lipid-lowering drugs revealed variations in their effect in modulating the morphological features of cholesterol particles.

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Genetics

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
Behnom Farboud *1,2, Erin Jarvis *1, Theodore L. Roth *3,4,5,6, Jiyung Shin *1,3, Jacob E. Corn 1,3, Alexander Marson 3,5,6,7,8,9, Barbara J. Meyer 1,2, Nipam H. Patel 1,10, Megan L. Hochstrasser 3
1Department of Molecular Cell Biology, University of California, Berkeley, 2Howard Hughes Medical Institute, University of California, Berkeley, 3Innovative Genomics Institute, University of California, Berkeley, 4Biomedical Sciences Graduate Program, University of California, San Francisco, 5Department of Microbiology and Immunology, University of California, San Francisco, 6Diabetes Center, University of California, San Francisco, 7Chan Zuckerberg Biohub, 8Department of Medicine, University of California, San Francisco, 9UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, 10Department of Integrative Biology, University of California, Berkeley

Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model organisms.

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Bioengineering

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing
Benjamin Demaree 1,2, Daniel Weisgerber 1, Freeman Lan 1,2, Adam R. Abate 1,2,3
1Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biosciences, University of California, San Francisco, 2UC Berkeley-UCSF Graduate Program in Bioengineering, University of California, San Francisco, 3Chan Zuckerberg Biohub

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing >50,000 single-cell genomes from diverse cell populations.

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Medicine

Cone Beam Intraoperative Computed Tomography-based Image Guidance for Minimally Invasive Transforaminal Interbody Fusion
Michael Safaee 1, Taemin Oh 1, Murat Pekmezci 2, Aaron J. Clark 1
1Department of Neurological Surgery, University of California, San Francisco, 2Department of Orthopedic Surgery, University of California, San Francisco

The purpose of this article is to provide image-guidance for minimally invasive transforaminal interbody fusion.

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Chemistry

Methionine Functionalized Biocompatible Block Copolymers for Targeted Plasmid DNA Delivery
Yang Wu *1, Wei Zhang *2, Jian Zhang 2, Zhi-Xiang Mao 3, Li Ding 2, Hao Li 3, Rong Ma 1, Jin-Hai Tang 2
1Research Center of Clinical Oncology, Jiangsu Cancer Hospital & Jiangsu Institute of Cancer Research & Nanjing Medical University Affiliated Cancer Hospital, 2Department of General Surgery, The First Affiliated Hospital with Nanjing Medical University, 3School of Clinical Medicine, Xuzhou Medical University

This work presents the preparation of methionine functionalized biocompatible block copolymers (mBG) via the reversible addition-fragmentation chain transfer (RAFT) method. The plasmid DNA complexing ability of the obtained mBG and their transfection efficiency were also investigated. The RAFT method is very beneficial for polymerizing monomers containing special functional groups.

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Cancer Research

Establishing Cell Lines Overexpressing DR3 to Assess the Apoptotic Response to Anti-mitotic Therapeutics
Xin Wang  *1, Jiamin Zhou *2, Chen Qi *1, Gelin Wang 1,3
1School of Pharmaceutical Sciences, Tsinghua University, 2Department of Radiology, University of California, San Francisco, 3Beijing Advanced Innovation Center for Structural Biology

Establishing a stable cell line overexpressing a gene of interest to study gene function can be done by stable transfection-picking single clones after transfecting them via retroviral infection. Here we show that HT29-DR3 cell lines generated in this way elucidate the mechanisms by which death receptor 3 (DR3) contributes to antimitotics-induced apoptosis.

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Immunology and Infection

Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation
Xiang Zhao 1, Maryam Hamidinia 1, Joanna Ai Ling Choo 1, Chien Tei Too 1,2, Zi Zong Ho 3, Ee Chee Ren 4, Antonio Bertoletti 3, Paul A. MacAry 1,2,5, Keith G. Gould 6, Joanna Brzostek 1, Nicholas R.J. Gascoigne 1,2,5
1Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, 2Immunology Programme, Life Sciences Institute, National University of Singapore, 3Emerging Infectious Diseases Program, Duke-NUS Graduate Medical School, 4Singapore Immunology Network, A*STAR, 5NUS Graduate School for Integrative Sciences and Engineering (NGS), National University of Singapore, 6Department of Immunology, Wright-Fleming Institute, Imperial College London

This protocol describes the use of single chain MHC class I complexes to investigate molecular interactions in human CD8+ T cell activation: generation of engineered antigen presenting cells expressing single chain constructs, culture of human CD8+ T cell clone and T cell activation experiments.

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Neuroscience

Convection Enhanced Delivery of Optogenetic Adeno-associated Viral Vector to the Cortex of Rhesus Macaque Under Guidance of Online MRI Images
Karam Khateeb 1,2, Devon J. Griggs 2,3, Philip N. Sabes 4, Azadeh Yazdan-Shahmorad 1,2,3,4
1Departments of Bioengineering, University of Washington, 2Washington National Primate Research Center, 3Department of Electrical and Computer Engineering, University of Washington, 4Department of Physiology and Center for Integrative Neuroscience, University of California, San Francisco

Here, we demonstrate magnetic resonance (MR)-guided convection enhanced delivery (CED) of viral vectors into the cortex as an efficient and simplified approach for achieving optogenetic expression across large cortical areas in the macaque brain.

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Cancer Research

Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment
Lei Wang *1, Huan Chen *2,3, Fei Fei *1, Xianfeng He 2,3, Shaoyang Sun 1, Kunpeng Lv 1, Bo Yu 2,3, Jiang Long 2,4,5,6, Xu Wang 1
1Department of Biochemistry and Molecular Biology, Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, School of Basic Medical Sciences, Fudan University, 2National Human Genetic Resources Sharing Service Platform (2005DKA21300), 3Fudan University Shanghai Cancer Center, 4Department of Pancreatic Surgery, Fudan University Shanghai Cancer Center, 5Department of Oncology, Shanghai Medical College, Fudan University, 6Pancreatic Cancer Institute, Fudan University

This protocol describes optimization procedures in a virus-based dual fluorescence-labeled tumor xenograft model using larval zebrafish as hosts. This heterogeneous xenograft model mimics the tissue composition of pancreatic cancer microenvironment in vivo and serves as a more precise tool for assessing drug responses in personalized zPDX (zebrafish patient-derived xenograft) models.

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Medicine

A Novel Non-invasive Method for the Detection of Elevated Intra-compartmental Pressures of the Leg
Matthew J. Herring 1, Erin Donohoe 1, Meir T. Marmor 1
1Department of Orthopedic Surgery, University of California, San Francisco

An ultrasound probe coupled with a pressure sensor is used to assess the intra-compartmental pressure of the leg by directly measuring the compartment fascia flattening pressure (CFFP). This non-invasive protocol will provide reliable assessment of the pressure inside the anterior muscular compartment of the lower leg.

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Cancer Research

In Vitro Assay to Study Tumor-macrophage Interaction
Zhenyi An 1, William A. Weiss 1,2,3
1Department of Neurology, University of California, San Francisco, 2Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, 3Departments of Pediatrics and Neurological Surgery, University of California, San Francisco

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

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Immunology and Infection

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization
Jessica N. Witchley 1, Pallavi M. Penumetcha 1, Suzanne M. Noble 1,2
1Department of Microbiology and Immunology, University of California, San Francisco, 2Department of Medicine, Division of Infectious Disease, University of California, San Francisco

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

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Developmental Biology

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development
Renat N. Khaliullin 1,2,3, Jeffrey M. Hendel 1,2, Adina Gerson-Gurwitz 1,2, Shaohe Wang 1,4,5, Stacy D. Ochoa 1,6, Zhiling Zhao 1,7, Arshad Desai 1,2, Karen Oegema 1,2, Rebecca A. Green 1,2
1Ludwig Institute for Cancer Research, San Diego, 2Department of Cellular and Molecular Medicine, University of California, San Diego, 3Recursion Pharmaceuticals, 4Biomedical Sciences Graduate Program, University of California, San Diego, 5Cell Biology Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, 6Department of Biology, San Diego State University, 7Developmental and Stem Cell Biology Graduate Program, University of California, San Francisco

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80–100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

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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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Bioengineering

A MRI-Based Toolbox for Neurosurgical Planning in Nonhuman Primates
William K.S. Ojemann 1,2, Devon J. Griggs 2,3, Zachary Ip 1,2, Olivya Caballero 4, Hesamoddin Jahanian 5, Susana Martinez-Conde 4, Stephen Macknik 4, Azadeh Yazdan-Shahmorad 1,2,3
1Bioengineering Department, University of Washington, 2Washington National Primate Research Center, University of Washington, 3Electrical and Computer Engineering Department, University of Washington, 4Department of Ophthalmology, SUNY Downstate Health Sciences University, 5Radiology Department, University of Washington

The method outlined below aims to provide a comprehensive protocol for the preparation of nonhuman primate (NHP) neurosurgery using a novel combination of three-dimensional (3D) printing methods and MRI data extraction.

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Automated Synthesis and Uptake Analysis of D-[Methyl-11C]-Methionine
Matthew F. L. Parker 1, Joseph E. Blecha 1, Brailee Schulte 1, Justin M. Luu 1, Megan N. Stewart 1, Robert R. Flavell 1, Henry F. VanBrocklin 1, Oren S. Rosenberg 2, Michael A. Ohliger 1, David M. Wilson 1
1Department of Radiology and Biomedical Imaging, University of California, San Francisco, 2Department of Medicine, University of California, San Francisco

Here, we present a protocol for the synthesis of D-[methyl-11C]-methionine, a metabolic positron emission tomography tracer for bacteria, and in vitro evaluation in Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. The synthesis involves a single synthetic step on an automated module, followed by microcentrifuge filtration.

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Neuroscience

Heterologous Expression and Functional Analysis of Aedes aegypti Odorant Receptors to Human Odors in Xenopus Oocytes
Xin Wang *1, Zhou Chen *1,2, Yifan Wang 1, Feng Liu 1,3, Shanshan Jiang 1, Nannan Liu 1
1Department of Entomology and Plant Pathology, Auburn University, 2Departments of Biochemistry & Biophysics, and Cellular & Molecular Pharmacology, University of California, San Francisco, 3Department of Biological Sciences, Vanderbilt University

A protocol is presented that functionally characterizes mosquito ORs in response to human odors using a Xenopus oocyte expression system coupled with a two-electrode voltage clamp, providing a powerful new technique for exploring the responses of mosquitoes ORs to exposure to human odors.

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Bioengineering

Simple, Affordable, and Modular Patterning of Cells using DNA
Katelyn A. Cabral 1, David M. Patterson 2, Olivia J. Scheideler 1, Russell Cole 3, Adam R. Abate 4,5,6, David V. Schaffer 7,8, Lydia L. Sohn 9, Zev J. Gartner 2,6,10
1Graduate Program in Bioengineering, University of California San Francisco and University of California Berkeley, 2Department of Pharmaceutical Chemistry, University of California San Francisco, 3Scribe Biosciences, 4Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, 5California Institute for Quantitative Biosciences, University of California San Francisco, 6Chan Zuckerberg Biohub, University of California San Francisco, 7Department of Chemical & Biomolecular Engineering, University of California Berkeley, 8Helen Wills Neuroscience Institute, University of California Berkeley, 9Department of Mechanical Engineering, University of California Berkeley, 10Center for Cellular Construction, University of California San Francisco

Here we present a protocol to micropattern cells at single-cell resolution using DNA-programmed adhesion. This protocol uses a benchtop photolithography platform to create patterns of DNA oligonucleotides on a glass slide and then labels cell membranes with commercially available complementary oligonucleotides. Hybridization of the oligos results in programmed cell adhesion.

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Biology

Recording Electrical Currents across the Plasma Membrane of Mammalian Sperm Cells
Boheng Liu 1,2, Nadine Mundt 3,4, Melissa Miller 1, David E. Clapham 5, Yuriy Kirichok 6, Polina V. Lishko 1
1Department of Molecular and Cell Biology, University of California, Berkeley, 2Department of Neurobiology, Peking University, 3Institute for Biology II / Chemosensation Lab, RWTH Aachen University, 4Research Training Group 2416 MultiSenses-MultiScales, RWTH Aachen University, 5Howard Hughes Medical Institute, Janelia Research Campus, 6Department of Physiology, University of California, San Francisco

This protocol describes how to perform electrical recordings from mammalian sperm cells in a whole-cell configuration, with the goal of directly recording ion channel activity. The method has been instrumental in describing the electrophysiological profiles of several sperm ion channels and helped to reveal their molecular identity and regulation.

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Biology

Particle Templated Emulsification enables Microfluidic-Free Droplet Assays
Daniel W. Weisgerber 1, Makiko N. Hatori 1, Adam R. Abate 1,2
1Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biosciences, University of California, 2Chan Zuckerberg Biohub

Water-in-oil droplet assays are useful for analytical chemistry, enzyme evolution, and single cell analysis, but typically require microfluidics to form the droplets. Here, we describe particle templated emulsification, a microfluidic-free approach to perform droplet assays.

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Cancer Research

Profiling Sensitivity to Targeted Therapies in EGFR-Mutant NSCLC Patient-Derived Organoids
Dora Barbosa Rabago 1,2,3, Collin M. Blakely 1,2, Franziska Haderk 1,2,3, Trever G. Bivona 1,2,3
1Department of Medicine, University of California, San Francisco, 2Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, 3Department of Cellular and Molecular Pharmacology, University of California, San Francisco

This protocol describes a standardized evaluation of drug sensitivities to targeted signaling inhibitors in NSCLC patient-derived organoid models.

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Immunology and Infection

Isolation of Primary Mouse Lung Endothelial Cells
Erika Wong 1,2, Nina Nguyen 2, Judith Hellman 2
1Department of Pediatrics, Division of Critical Care, UCSF Benioff Children's Hospital, 2Department of Anesthesia and Perioperative Care, University of California, San Francisco

In this article, primary lung endothelial cells were isolated and cultured from neonatal mice.

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Medicine

A Modified Vessel-Sparing Microsurgical Vasoepididymostomy
Liang Zhou 1, Xingzhe Ji 1, Lei Wang 1
1Assisted Reproduction Center, Northwest Women’s and Children’s Hospital

Here, we present a protocol to preserve the vasal vessels in microsurgical vasoepididymostomy. The surgical security is enhanced by preserving the vasal vessels using a retrograde-anterograde dissociation and fixing the vasal vessels.

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Immunology and Infection

A Mouse Model of Orotracheal Intubation and Ventilated Lung Ischemia Reperfusion Surgery
Wen-I Liao *1, Daisuke Maruyama *1, Farzaneh Kianian 1,2, Christine Tat 1, Xiaoli Tian 1, Judith Hellman 1, Jeffrey M Dodd-o 3, Arun Prakash 1
1Department of Anesthesia and Perioperative Care, University of California San Francisco and San Francisco General Hospital, 2Department of Physiology, School of Medicine, Tehran University of Medical Sciences, 3Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine

A mouse surgical model to create left lung ischemia reperfusion (IR) injury while maintaining ventilation and avoiding hypoxia.

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Biochemistry

"Cell Surface Capture" Workflow for Label-Free Quantification of the Cell Surface Proteome
Akul Naik 1, Sanjeeva Srivastava 1,2, Arun P. Wiita 1,3,4
1Department of Laboratory Medicine, University of California, San Francisco, 2Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, 3Deptartment of Bioengineering and Therapeutic Sciences, University of California, San Francisco, 4Chan Zuckerberg Biohub

Here, we describe a proteomics workflow for characterization of the cell surface proteome of various cell types. This workflow includes cell surface protein enrichment, subsequent sample preparation, analysis using an LC-MS/MS platform, and data processing with specialized software.

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Methods For Visualizing Intracellular Organelles
Xiang Zhao 1,2, Su Guo 1,2,3
1Chan Zuckerberg Biohub, 2Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, 3Programs in Human Genetics and Biological Sciences, University of California, San Francisco

Methods For Visualizing Intracellular Organelles

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Neuroscience

Closed-Loop Neurostimulation for Biomarker-Driven, Personalized Treatment of Major Depressive Disorder
Kristin K. Sellers 1,2, Ankit N. Khambhati 1,2, Noah Stapper 2,3, Joline M. Fan 2,4, Vikram R. Rao 2,4, Katherine W. Scangos 2,3, Edward F. Chang 1,2, Andrew D. Krystal 2,3
1Department of Neurological Surgery, University of California, San Francisco, 2Weill Institute for Neurosciences, University of California, San Francisco, 3Department of Psychiatry and Behavioral Sciences, University of California, San Francisco, 4Department of Neurology, University of California, San Francisco

Deep brain stimulation triggered by a patient-specific neural biomarker of a high-symptom state may better control symptoms of major depressive disorder compared to continuous, open-loop stimulation. This protocol provides a workflow for identifying a patient-specific neural biomarker and controlling the delivery of therapeutic stimulation based on the identified biomarker.

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Neuroscience

Bringing the Clinic Home: An At-Home Multi-Modal Data Collection Ecosystem to Support Adaptive Deep Brain Stimulation
Gabrielle Strandquist 1, Tomasz Frączek 2, Tanner Dixon 3, Shravanan Ravi 3, Raphael Bechtold 4, Daryl Lawrence 5, Alicia Zeng 6, Jack Gallant 7, Simon Little 3, Jeffrey Herron 8
1Computer Science and Engineering, University of Washington, 2Neuroscience, University of Washington, 3Neurology, University of California, San Francisco, 4Bioengineering, University of Washington, 5Bioengineering, University of California, Berkeley, 6Biophysics, University of California, Berkeley, 7Psychology, University of California, Berkeley, 8Neurological Surgery, University of Washington

The protocol shows a prototype of the at-home multi-modal data collection platform that supports research optimizing adaptive deep brain stimulation (aDBS) for people with neurological movement disorders. We also present key findings from deploying the platform for over a year to the home of an individual with Parkinson's disease.

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JoVE Core

Quantifying Pain Location and Intensity with Multimodal Pain Body Diagrams
Jereen Kwong *1, Joanna Lin *2, Ryan Leriche *2, Thomas A. Wozny 2, Ana Shaughnessy 2, Ashlyn Schmitgen 2, Prasad Shirvalkar 3
1Anesthesiology (Division of Pain Management), University of California, San Francisco, 2Neurological Surgery and UCSF Weill Institute of Neurosciences, University of California, San Francisco, 3Anesthesiology (Division of Pain Management) and Neurological Surgery and UCSF Weill Institute of Neurosciences, University of California, San Francisco

Current pain scales used to quantify pain severity, such as visual analog scales, fail to capture the complexity of subjective pain experiences. Pain body diagrams are qualitative but may be more informative. The goal of this method is to extract quantitative metrics from pain body diagrams using novel pressure-hue transformation.

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Medicine

Erosion Identification in Metacarpophalangeal Joints in Rheumatoid Arthritis using High-Resolution Peripheral Quantitative Computed Tomography
Yousif Al-Khoury 1,2, Stephanie Finzel 3, Camille Figueiredo 4, Andrew J. Burghardt 5, Kathryn S. Stok 6, Lai-Shan Tam 7, Isaac Cheng 7, Justin J. Tse 1, Sarah L. Manske 1
1Department of Radiology, McCaig Institute for Bone and Joint Health, University of Calgary, 2Department of Biomedical Engineering, University of Calgary, 3University Medical Center Freiburg, 4Department of Rheumatology, University of São Paulo, 5Department of Radiology and Biomedical Imaging, University of California, San Francisco, 6Department of Biomedical Engineering, University of Melbourne, 7Department of Medicine and Therapeutics, The Chinese University of Hong Kong

Bone erosions are an important pathological feature of rheumatoid arthritis. The purpose of this work is to introduce a training tool to provide users with guidance on identifying pathological cortical breaks on high resolution peripheral quantitative computed tomography images for erosion analysis.

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Neuroscience

Differentiation of Enteric Nervous System Lineages from Human Pluripotent Stem Cells
Homa Majd 1,2, Mikayla N. Richter 1,2, Ryan M. Samuel 1,2, Ali Kalantari 1,2, Jonathan T. Ramirez 1,2, Faranak Fattahi 1,2,3
1Department of Cellular and Molecular Pharmacology, University of California, San Francisco, 2Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, 3Program in Craniofacial Biology, University of California, San Francisco

Deriving enteric nervous system (ENS) lineages from human pluripotent stem cells (hPSC) provides a scalable source of cells to study ENS development and disease, and to use in regenerative medicine. Here, a detailed in vitro protocol to derive enteric neurons from hPSCs using chemically defined culture conditions is presented.

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Medicine

Surgical Bone Implantation Technique for Rat Tibia Models of Diabetes and Osteoporosis
David Alfaiate 1,2, Carlos Mas-Moruno 3,4, Cidália Manuela 5, Josep Maria Ustrell 6,7, Juan Antonio Camara 8,9, Marina Ferrer 10,11, José Maria Manero 3,4, Maria-Cristina Manzanares-Céspedes 12
1Faculty of Medicine and Health Sciences, University of Barcelona, 2Department of Oral Surgery, Davallmed Clinic. Porto, 3Biomaterials, Biomechanics and Tissue Engineering Group, Department of Materials Science and Engineering, Universitat Politècnica de Catalunya, 4Barcelona Research Centre in Multiscale Science and Engineering, Universitat Politècnica de Catalunya, 5Department of Periodontology, Davallmed Clinic. Porto, 6Department of Odontostomatology, University of Barcelona, 7Oral Health and Masticatory System Research Group at the Bellvitge Biomedical Research Institute (IDIBELL), 8Preclinical imaging platform, Vall d'Hebron Research Institute, Barcelona, 9Preclinical Therapeutics Core, Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, 10Animal facility, Vall d'Hebron Research Institute, Barcelona, 11UCSF Gnotobiotics Core Facility, University of California, San Francisco, 12Human Anatomy and Embryology Unit, Experimental Pathology and Therapeutics Department, Faculty of Medicine and Health Sciences, University of Barcelona

The placement of implants in a rat model is an essential experimental procedure for clinical research. This study presents a comprehensive surgical protocol for implanting titanium implants into the tibia of rat models with diabetes and osteoporosis.

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Neuroscience

Monitoring Blood-Brain Barrier Opening in Rats with a Preclinical Focused Ultrasound System
Kisoo Kim 1,2, Marco Gallus 3, Tianrun Xiao 1, Akshay S. Parchure 4, Bhavya R. Shah 4, Hideho Okada 3, Chris Diederich 5, Eugene Ozhinsky *1, Kazim Narsinh *1
1Department of Radiology & Biomedical Imaging, University of California, San Francisco, 2Department of Biomedical Engineering, Kyung Hee University, 3Department of Neurological Surgery, University of California, San Francisco, 4University of Texas Southwestern Medical Center, 5Department of Radiation Oncology, University of California, San Francisco

This study describes a combined magnetic resonance imaging (MRI) and low-intensity pulsed focused ultrasound (FUS) protocol, utilizing living rats with jugular vein catheterization to monitor blood-brain barrier (BBB) opening.

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