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32 ARTICLES PUBLISHED IN JoVE

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Biology

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes
Eiji Shigetomi 1, Baljit S. Khakh 1
1Departments of Physiology and Neurobiology, David Geffen School of Medicine, University of California, Los Angeles

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

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Biology

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels
Harpreet Singh 1, Sarah Warburton 2, Thomas M. Vondriska 3, Baljit S. Khakh 1
1Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, 2Department of Anesthesiology, David Geffen School of Medicine, University of California, Los Angeles, 3Department of Anesthesiology, Medicine and Physiology, David Geffen School of Medicine, University of California, Los Angeles

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

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Behavior

Method for Simultaneous fMRI/EEG Data Collection during a Focused Attention Suggestion for Differential Thermal Sensation
Pamela K. Douglas 1,2, Maureen Pisani 2, Rory Reid 1, Austin Head 2, Edward Lau 2, Ebrahim Mirakhor 3, Jennifer Bramen 2, Billi Gordon 2, Ariana Anderson 2, Wesley T. Kerr 2, Chajoon Cheong 4, Mark S. Cohen 1,2
1Neuropsychiatric Institute, University of California, Los Angeles, 2Laboratory of Neuroimaging Technology, University of California, Los Angeles, 3Yale School of Medicine, 4Korean Basic Science Institute

We present a protocol for concurrent collection of EEG/fMRI data, and synchronized MR clock signal recording. We demonstrate this method using a unique paradigm whereby subjects receive ‘cold glove’ instructions during scanning, and EEG/fMRI data are recorded along with hand temperature measurements both before and after hypnotic induction.

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Bioengineering

Bacterial Detection & Identification Using Electrochemical Sensors
Colin Halford 1,2, Vincent Gau 3, Bernard M. Churchill 2, David A. Haake 2,4,5
1Research Service, Veterans Affairs Greater Los Angeles Healthcare System, 2Department of Urology, The David Geffen School of Medicine, University of California, Los Angeles , 3GeneFluidics, 4Division of Infectious Diseases, Veterans Affairs Greater Los Angeles Healthcare System, 5Department of Microbiology, Immunology & Molecular Genetics, University of California, Los Angeles

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

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Immunology and Infection

Bioluminescent Bacterial Imaging In Vivo
Chwanrow K. Baban *1, Michelle Cronin *1, Ali R. Akin 1, Anne O'Brien 1, Xuefeng Gao 1, Sabin Tabirca 1, Kevin P. Francis 1, Mark Tangney 1
1Cork Cancer Research Centre, BioSciences Institute, University College Cork

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

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Medicine

Network Analysis of the Default Mode Network Using Functional Connectivity MRI in Temporal Lobe Epilepsy
Zulfi Haneef 1,2, Agatha Lenartowicz 3, Hsiang J. Yeh 4, Jerome Engel Jr. 4, John M. Stern 4
1Department of Neurology, Baylor College of Medicine, 2Neurology Care Line, Michael E. DeBakey VA Medical Center, 3Semel Institute for Neuroscience and Human Behavior, University of California, Los Angeles, 4Department of Neurology, University of California, Los Angeles

The Default Mode Network (DMN) in Temporal Lobe Epilepsy (TLE) is analyzed in the resting state of the brain using seed-based functional connectivity MRI (fcMRI).

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Medicine

Combined In vivo Optical and µCT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice
Nicholas M. Bernthal 1, Brad N. Taylor 2, Jeffrey A. Meganck 2, Yu Wang 3, Jonathan H. Shahbazian 3, Jared A. Niska 1, Kevin P. Francis 2, Lloyd S. Miller 3,4
1Orthopaedic Hospital Research Center, Orthopaedic Hospital Department of Orthopaedic Surgery, David Geffen School of Medicine at University of California, Los Angeles (UCLA), 2PerkinElmer, 3Department of Dermatology, Johns Hopkins University School of Medicine, 4Department of Medicine, Division of Infectious Diseases, Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine

Combined optical and μCT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

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Neuroscience

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators
Ruotian Jiang 1, Martin D. Haustein 1, Michael V. Sofroniew 2, Baljit S. Khakh 1,2
1Department of Physiology, University of California Los Angeles, 2Department of Neurobiology, University of California Los Angeles

The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ.

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Developmental Biology

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans
Daniela A. Parodi 1, Robert Damoiseaux 3, Patrick Allard 1,2
1Institute for Society and Genetics, University of California, Los Angeles, 2Environmental Health Sciences, University of California, Los Angeles, 3California Nanosystems Institute, Department of Molecular and Medical Pharmacology, University of California, Los Angeles

We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

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Bioengineering

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts
Natalie L. Leong 1, Nima Kabir 1, Armin Arshi 1, Azadeh Nazemi 2, Ben M. Wu 2, David R. McAllister 1, Frank A. Petrigliano 1
1Department of Orthopaedic Surgery, University of California Los Angeles, 2Department of Bioengineering, University of California Los Angeles

Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

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Behavior

Measurement of Neurophysiological Signals of Ignoring and Attending Processes in Attention Control
Agatha Lenartowicz 1, Gregory V. Simpson 2, Samantha R. O'Connell 1, Mark S. Cohen 3
1Department of Psychiatry, University of California Los Angeles, 2Attention Research Institute, 3Departments of Psychiatry, Radiology, Neurology, Biomedical Physics, Psychology and Bioengineering, University of California Los Angeles

Attention control comprises enhancement of target signals and attenuation of distractor signals. We describe an approach to measure separately but concurrently, the neurophysiology of attending and ignoring in sustained intermodal attention, utilizing a passive control condition during which neither process is continuously engaged.

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Medicine

A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology
Stefanie Nunez 1, M. Mehdi Doroudchi 1, Amy J. Gleichman 1, Kwan L. Ng 1, Irene L. Llorente 1, Elif G. Sozmen 1, S. Thomas Carmichael 1, Jason D. Hinman 1
1Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles

Here we present methodology for the production of a focal stroke in murine white matter by local injection of an irreversible endothelial nitric oxide synthase (eNOS) inhibitor (L-Nio). Presented are two stereotactic variations, retrograde neuronal tracing, and fresh tissue labeling and dissection that expand the potential applications of this technique.

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Immunology and Infection

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection
Kristel Lourdault 1,2, James Matsunaga 1,2, Karen V. Evangelista 1,2, David A. Haake 1,2,3,4
1Veterans Affairs Greater Los Angeles Healthcare System, 2Departments of Medicine, David Geffen School of Medicine at University of California Los Angeles, 3Departments of Urology, David Geffen School of Medicine at University of California Los Angeles, 4Departments of Microbiology, Immunology, and Molecular Genetics, University of California Los Angeles

We describe here a technique that combines transposon mutagenesis with high-throughput sequencing to identify and quantify transposon leptospiral mutants in tissues after a challenge of hamsters. This protocol can be used to screen mutants for survival and dissemination in animals and can also be applied to in vitro studies.

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Cancer Research

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer
Milica Momcilovic 1, Sean T. Bailey 2, Jason T. Lee 3, Charles Zamilpa 3, Anthony Jones 3, Gihad Abdelhady 1, James Mansfield 4, Kevin P. Francis 5, David B. Shackelford 1
1Division of Pulmonary and Critical Care Medicine, University of California Los Angeles David Geffen School of Medicine, 2University of North Carolina at Chapel Hill, 3Department of Molecular and Medical Pharmacology, University of California Los Angeles, 4Andor Technology, 5Division of Orthopaedic Surgery, University of California Los Angeles David Geffen School of Medicine

In this protocol, we describe how to utilize [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography (18F-FDG PET/CT) imaging to measure the tumor metabolic response to the targeted therapy MLN0128 in a Kras/Lkb1 mutant mouse model of lung cancer and coupled imaging with high resolution ex vivo autoradiography and quantitative histology.

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Neuroscience

Making, Testing, and Using Potassium Ion Selective Microelectrodes in Tissue Slices of Adult Brain
J. Christopher Octeau 1, Guido Faas 2, Istvan Mody 1,2, Baljit S. Khakh 1,3
1Department of Physiology, David Geffen School of Medicine, University of California Los Angeles, 2Department of Neurology, David Geffen School of Medicine, University of California Los Angeles, 3Department of Neurobiology, David Geffen School of Medicine, University of California Los Angeles

Potassium ions contribute to the resting membrane potential of cells and extracellular K+ concentration is a crucial regulator of cellular excitability. We describe how to make, calibrate and use monopolar K+-selective microelectrodes. Using such electrodes enables the measurement of electrically evoked K+ concentration dynamics in adult hippocampal slices.

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Bioengineering

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart
Victoria Messerschmidt *1, Zachary Bailey *1, Kyung In Baek 2, Yichen Ding 2, Jeffrey J. Hsu 2, Richard Bryant 1, Rongsong Li 3, Tzung K. Hsiai 2, Juhyun Lee 1
1Department of Bioengineering, The University of Texas at Arlington, 2Department of Medicine (Cardiology) and Bioengineering, UCLA, 3College of Health Science and Environmental Engineering, Shenzhen Technology University

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

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Bioengineering

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart
Yichen Ding 1, Zachary Bailey 2, Victoria Messerschmidt 2, Jun Nie 3, Richard Bryant 2, Sandra Rugonyi 4, Peng Fei 3, Juhyun Lee 1,2, Tzung K. Hsiai 1
1Department of Bioengineering, University of California Los Angeles, 2Department of Bioengineering, University of Texas at Arlington, 3School of Optical and Electronic Information, Huazhong University of Science and Technology, 4Department of Biomedical Engineering, OSHU

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

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Bioengineering

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells
Weikun Xiao 1, Arshia Ehsanipour 1, Alireza Sohrabi 1, Stephanie K. Seidlits 2
1Department of Bioengineering, University of California, Los Angeles, 2Department of Bioengineering, Jonsson Comprehensive Cancer Center, Broad Stem Cell Research Center, Brain Research Institute, University of California, Los Angeles

Here, we present a protocol for three-dimensional culture of patient-derived glioblastoma cells within orthogonally tunable biomaterials designed to mimic the brain matrix. This approach provides an in vitro, experimental platform that maintains many characteristics of in vivo glioblastoma cells typically lost in culture.

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Medicine

Use of MRI-ultrasound Fusion to Achieve Targeted Prostate Biopsy
Rajiv Jayadevan 1, Steve Zhou 1, Alan M. Priester 1, Merdie Delfin 1, Leonard S. Marks 1
1Department of Urology, University of California Los Angeles

Presented herein is a protocol to perform targeted biopsy of the prostate using an MRI-ultrasound fusion system.

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Neuroscience

Visualizing Astrocyte Morphology Using Lucifer Yellow Iontophoresis
Stefanie L. Moye 1, Blanca Diaz-Castro 1, Mohitkumar R. Gangwani 1, Baljit S. Khakh 1,2
1Department of Physiology, David Geffen School of Medicine, University of California Los Angeles, 2Department of Neurobiology, David Geffen School of Medicine, University of California Los Angeles

Astrocytes are morphologically complex cells, exemplified by their multiple processes and bushy territories. To analyze their elaborate morphology, we present a reliable protocol to perform intracellular Lucifer yellow iontophoresis in lightly fixed tissue.

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Cancer Research

Comparing Metastatic Clear Cell Renal Cell Carcinoma Model Established in Mouse Kidney and on Chicken Chorioallantoic Membrane
Moe Ishihara *1, Junhui Hu *1, Xiaoyu Zhang 2, YongHyeon Choi 3, Anthony Wong 4, Celine Cano-Ruiz 5, Rongwei Zhao 6, Ping Tan 7, Jonathan L. Tso 1, Lily Wu 1,8
1Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, 2Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, 3Department of Bioengineering, Hanyang University, 4Department of Ecology and Evolutionary Biology, University of California, Los Angeles, 5Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, 6School of Life Sciences, Beijing Normal University, 7Department of Urology, West China Hospital, Sichuan University, 8Department of Urology, David Geffen School of Medicine, University of California, Los Angeles

Metastatic clear cell renal cell carcinoma is a disease without a comprehensive animal model for thorough preclinical investigation. This protocol illustrates two novel animal models for the disease: the orthotopically implanted mouse model and the chicken chorioallantoic membrane model, both of which demonstrate lung metastasis resembling clinical cases.

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Medicine

In Vivo Mouse Model of Spinal Implant Infection
Benjamin V. Kelley 1, Christopher Hamad 1, Stephen D. Zoller 1, Danielle Greig 1, Zeinab Mamouei 1, Rene Chun 1, Kellyn Hori 1, Nicolas Cevallos 1, Chad Ishmael 1, Peter Hsiue 1, Rishi Trikha 1, Troy Sekimura 2, Brandon Gettleman 3, Autreen Golzar 2, Adrian Lin 2, Thomas Olson 2, Ameen Chaudry 2, Michael M. Le 2, Anthony A. Scaduto 1, Kevin P. Francis 1, Nicholas M. Bernthal 1
1Department of Orthopaedic Surgery, University of California Los Angeles, 2David Geffen School of Medicine, University of California Los Angeles, 3University of South Carolina School of Medicine, University of South Carolina

The protocol describes a novel in vivo mouse model of spinal implant infection where a stainless-steel k-wire implant is infected with bioluminescent Staphylococcus aureus Xen36. Bacterial burden is monitored longitudinally with bioluminescent imaging and confirmed with colony forming unit counts after euthanasia.

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Cancer Research

Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers
Allison C. Sharrow 1, Moe Ishihara 1, Junhui Hu 1, Il Hyun Kim 1, Lily Wu 1
1Molecular and Medical Pharmacology, University of California Los Angeles

We present the chicken chorioallantoic membrane model as an alternative, transplantable, in vivo model for the engraftment of gynecological and urological cancer cell lines and patient-derived tumors.

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Medicine

Novel Percutaneous Approach for Deployment of 3D Printed Coronary Stenosis Implants in Swine Models of Ischemic Heart Disease
John J. Hollowed 1,2, Caroline M. Colbert 3,4, Jesse W. Currier 1,2, Kim-Lien Nguyen 1,2,3,4
1Division of Cardiology, David Geffen School of Medicine, University of California Los Angeles, 2VA Greater Los Angeles Healthcare System, U.S. Department of Veterans Affairs, 3Physics and Biology in Medicine Graduate Program, University of California Los Angeles, 4Diagnostic Cardiovascular Imaging Laboratory, Department of Radiological Sciences, David Geffen School of Medicine, University of California Los Angeles

We describe a novel, cost-effective, and efficient technique for percutaneous delivery of three-dimensionally printed coronary implants to create closed-chest swine models of ischemic heart disease. The implants were fixed in place using a mother-and-child extension catheter with high success rate.

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Biology

Injection of Hydrogel Biomaterial Scaffolds to The Brain After Stroke
Katrina L. Wilson 1, S. Thomas Carmichael 2, Tatiana Segura 1,3,4
1Department of Biomedical Engineering, Duke University, 2Department of Neurology, University of California Los Angeles, 3Department of Neurology, Duke University, 4Department of Dermatology, Duke University

Stroke is a global issue with minimal treatment options and no current clinical therapy for regenerating the lost brain tissue. Here we describe methods for creating precise photothrombotic stroke in the motor cortex of rodents and subsequent injection of hydrogel biomaterials to study their effects on tissue regeneration after stroke.

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Medicine

Focal Laser Ablation of Prostate Cancer: An Office Procedure
Wayne G. Brisbane 1, Shyam Natarajan 1, Alan Priester 1, Ely R. Felker 1, Adam Kinnaird 2, Leonard S. Marks 1
1University of California Los Angeles, 2University of Alberta

This article presents and describes an outpatient treatment for prostate cancer using focal laser ablation. Laser catheter placement is guided by MRI-ultrasound fusion imaging in a fashion similar to prostate needle biopsy. Treatment is monitored in real-time with a thermal probe, placed adjacent to the laser fiber.

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Biochemistry

Microcrystal Electron Diffraction of Small Molecules
Michael W. Martynowycz 1,2, Tamir Gonen 1,2,3
1Howard Hughes Medical Institute, University of California Los Angeles, 2Department of Biological Chemistry, University of California Los Angeles, 3Department of Physiology, University of California Los Angeles

Here, we describe the procedures developed in our laboratory for preparing powders of small molecule crystals for microcrystal electron diffraction (MicroED) experiments.

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Biology

The Use of the Patch-Clamp Technique to Study the Thermogenic Capacity of Mitochondria
Ambre M. Bertholet 1
1Department of Physiology, David Geffen School of Medicine, University of California Los Angeles

This method article details the main steps in measuring H+ leak across the inner mitochondrial membrane with the patch-clamp technique, a new approach to study the thermogenic capacity of mitochondria.

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Biology

Planarian Ovary Dissection for Ultrastructural Analysis and Antibody Staining
Fengli Guo 1, Melainia McClain 1, Xia Zhao 1, Kexi Yi 1, Tari Parmely 1, Jay Unruh 1, Brian Slaughter 1, Leonid Kruglyak 2,3, Longhua Guo 2,3, Alejandro Sánchez Alvarado 1,2
1Stowers Institute for Medical Research, 2Howard Hughes Medical Institute, 3Department of Human Genetics, University of California Los Angeles

This protocol presents steps taken to dissect ovaries in the freshwater planarians, Schmidtea mediterranea. The dissected ovaries are compatible for antibody immunostaining and ultrastructural analysis with transmission electron microscopy to study the cell biology of the oocytes and somatic cells, providing an imaging depth and quality that were previously inaccessible.

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Bioengineering

Simplified, High-throughput Analysis of Single-cell Contractility using Micropatterned Elastomers
Lara Hairapetian 1, Enrico Cortes 1, Junyi Zhao 1, Yao Wang 1, Ricky Huang 1, Robert Damoiseaux 1,2, Ivan Pushkarsky 1
1Forcyte Biotechnologies, 2University of California, Los Angeles

This work presents a flexible protocol for utilizing fluorescently labeled elastomeric contractible surfaces (FLECS) Technology in microwell format for simplified, hands-off quantification of single-cell contractile forces based on visualized displacements of fluorescent protein micropatterns.

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Bioengineering

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models
Jesse Liang 1, Alireza Sohrabi 1, Mary Epperson 1, Laila M. Rad 1, Kelly Tamura 1, Mayilone Sathialingam 1, Thamira Skandakumar 1, Philip Lue 1, Jeremy Huang 1, James Popoli 1, Aidan Yackly 1, Michael Bick 1, Ze Zhong Wang 1, Chia-Chun Chen 1, Grigor Varuzhanyan 1, Robert Damoiseaux 1, Stephanie K. Seidlits 1
1University of California Los Angeles

The present protocol describes an experimental platform to assess the effects of mechanical and biochemical cues on chemotherapeutic responses of patient-derived glioblastoma cells in 3D matrix-mimetic cultures using a custom-made UV illumination device facilitating high-throughput photocrosslinking of hydrogels with tunable mechanical features.

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Developmental Biology

Using Ex Vivo Live Imaging to Investigate Cell Divisions and Movements During Mouse Dental Renewal
Abinaya Sundari Thooyamani *1, Elias Shahin *2, Sanako Takano 1, Amnon Sharir 2, Jimmy K. Hu 1,3
1School of Dentistry, University of California Los Angeles, 2The Institute of Biomedical and Oral Research, Faculty of Dental Medicine, Hebrew University of Jerusalem, 3Molecular Biology Institute, University of California Los Angeles

Ex vivo live imaging is a powerful technique for studying the dynamic processes of cellular movements and interactions in living tissues. Here, we present a protocol that implements two-photon microscopy to live track dental epithelial cells in cultured whole adult mouse incisors.

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