Princeton University View Institution's Website 30 articles published in JoVE Bioengineering 3D Printing Bacteria to Study Motility and Growth in Complex 3D Porous Media R. Kōnane Bay1,2, Anna M. Hancock2, Arabella S. Dill-Macky2, Hao Nghi Luu2, Sujit S. Datta2 1Department of Chemical and Biological Engineering, University of Colorado Boulder, 2Department of Chemical and Biological Engineering, Princeton University This protocol describes a procedure for three-dimensional (3D) printing of bacterial colonies to study their motility and growth in complex 3D porous hydrogel matrices that are more akin to their natural habitats than conventional liquid cultures or Petri dishes. Developmental Biology Optogenetic Inhibition of Rho1-Mediated Actomyosin Contractility Coupled with Measurement of Epithelial Tension in Drosophila Embryos Hanqing Guo1,2, Michael Swan3, Bing He1 1Department of Biological Sciences, Dartmouth College, 2School of Life Sciences, Westlake University, 3Department of Molecular Biology, Princeton University Actomyosin contractility plays an important role in cell and tissue morphogenesis. However, it is challenging to manipulate actomyosin contractility in vivo acutely. This protocol describes an optogenetic system that rapidly inhibits Rho1-mediated actomyosin contractility in Drosophila embryos, revealing the immediate loss of epithelial tension after the inactivation of actomyosin in vivo. Biology Sample Preparation for Rapid Lipid Analysis in Drosophila Brain Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Yuki X. Chen*1,5,6, Kelly Veerasammy*1,2, Jun Yin*3, Tenzin Choetso1,4, Tiffany Zhong7, Muniyat A. Choudhury1,4,5, Cory Weng1,4,5, Ethan Xu8, Mayan A. Hein9,10, Rinat Abzalimov2, Ye He1 1Advanced Science Research Center, Neuroscience Initiative, the City University of New York, Graduate Center New York, 2Advanced Science Research Center, Structural Biology Initiative, the City University of New York, Graduate Center, 3National Institute of Neurological Disorders and Stroke, National Institutes of Health, 4The City College of New York, CUNY, 5Macaulay Honors College, CUNY, 6Graduate School of Public Health and Health Policy, The City University of New York, 7Princeton University, 8Ardrey Kell High School, 9The Borough of Manhattan Community College, CUNY, 10Gallatin School of Individualized Study, New York University The aim of this protocol is to provide detailed guidance on the proper sample preparation for lipid and metabolite analysis in small tissues, such as the Drosophila brain, using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging. Bioengineering Light-Controlled Fermentations for Microbial Chemical and Protein Production Shannon M. Hoffman*1, Makoto A. Lalwani*1, José L. Avalos1,2,3,4 1Department of Chemical and Biological Engineering, Princeton University, 2The Andlinger Center for Energy and the Environment, Princeton University, 3Department of Molecular Biology, Princeton University, 4High Meadows Environmental Institute, Princeton University Optogenetic control of microbial metabolism offers flexible dynamic control over fermentation processes. The protocol here shows how to set up blue light-regulated fermentations for chemical and protein production at different volumetric scales. Neuroscience A Flexible Platform for Monitoring Cerebellum-Dependent Sensory Associative Learning Gerard Joey Broussard1, Mikhail Kislin1, Caroline Jung1, Samuel S. -H. Wang1 1Neuroscience Institute, Princeton University We have developed a single platform to track animal behavior during two climbing fiber-dependent associative learning tasks. The low-cost design allows integration with optogenetic or imaging experiments directed towards climbing fiber-associated cerebellar activity. Cancer Research A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth David Jelinek1,2, Ellen Ran Zhang1,2,3, Aaron Ambrus1,2, Erin Haley3, Emily Guinn1,2, Austin Vo1,2, Peter Le1,2, Ayse Elif Kesaf1,2, Jennifer Nguyen1,2, Lily Guo1,2, Destiny Frederick1,2, Zhengyang Sun1,2, Natalie Guo3, Parker Sevier1,2, Eric Bilotta1,2, Kaiser Atai1,2,4, Laurent Voisin1,2, Hilary A. Coller1,2,4 1Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, 2Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, 3Department of Molecular Biology, Princeton University, 4Molecular Biology Institute, University of California, Los Angeles A protocol to co-inject cancer cells and fibroblasts and monitor tumor growth over time is provided. This protocol can be used to understand the molecular basis for the role of fibroblasts as regulators of tumor growth. Immunology and Infection Mouse Footpad Inoculation Model to Study Viral-Induced Neuroinflammatory Responses Kathlyn Laval1, Carola J. Maturana2, Lynn W. Enquist1 1Department of Molecular Biology, Princeton University, 2Princeton Neuroscience Institute, Princeton University The footpad inoculation model is a valuable tool for characterizing viral-induced neuroinflammatory responses in vivo. In particular, it provides a clear assessment of viral kinetics and associated immunopathological processes initiated in the peripheral nervous system. Bioengineering Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior Francesco Carrara1, Douglas R. Brumley2, Andrew M. Hein3, Yutaka Yawata4,5, M. Mehdi Salek1, Kang Soo Lee1, Elzbieta Sliwerska1, Simon A. Levin6, Roman Stocker1 1Institute of Environmental Engineering, Department of Civil, Environmental and Geomatic Engineering, 2School of Mathematics and Statistics, University of Melbourne, 3Institute of Marine Sciences, University of California, Santa Cruz, 4Faculty of Life and Environmental Sciences, University of Tsukuba, 5Microbiology Research Center for Sustainability, University of Tsukuba, 6Department of Ecology and Evolutionary Biology, Princeton University A protocol for the generation of dynamic chemical landscapes by photolysis within microfluidic and millifluidic setups is presented. This methodology is suitable to study diverse biological processes, including the motile behavior, nutrient uptake, or adaptation to chemicals of microorganisms, both at the single cell and population level. Immunology and Infection Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization Benjamin P. Bratton2, Brody Barton1, Randy M. Morgenstein1 1Department of Microbiology and Molecular Genetics, Oklahoma State University, 2Department of Molecular Biology and Lewis-Sigler Institute of Integrative Genomics, Princeton University This protocol explains how to prepare and mount bacterial samples for live three-dimensional imaging and how to reconstruct the three-dimensional shape of E. coli from those images. Bioengineering Generation of Heterogeneous Drug Gradients Across Cancer Populations on a Microfluidic Evolution Accelerator for Real-Time Observation Ke-Chih Lin1, Gonzalo Torga2, Yusha Sun1, Kenneth J. Pienta2, James C. Sturm1, Robert H. Austin1 1Princeton University, 2Johns Hopkins Medical Institute We present a microfluidic cancer-on-chip model, the "Evolution Accelerator" technology, which provides a controllable platform for long-term real-time quantitative studies of cancer dynamics within well-defined environmental conditions at the single-cell level. This technology is expected to work as an in vitro model for fundamental research or pre-clinical drug development. Bioengineering Flash NanoPrecipitation for the Encapsulation of Hydrophobic and Hydrophilic Compounds in Polymeric Nanoparticles Chester E. Markwalter, Robert F. Pagels, Brian K. Wilson, Kurt D. Ristroph, Robert K. Prud'homme 1Department of Chemical and Biological Engineering, Princeton University Flash NanoPrecipitation (FNP) is a scalable approach to produce polymeric core-shell nanoparticles. Lab-scale formulations for the encapsulation of hydrophobic or hydrophilic therapeutics are described. Chemistry Preparation and Characterization of C60/Graphene Hybrid Nanostructures Chuanhui Chen1, Adam Mills1,2, Husong Zheng1, Yanlong Li1, Chenggang Tao1 1Department of Physics, Center for Soft Matter and Biological Physics, Virginia Tech, 2Department of Physics, Princeton University Here we present a protocol for the fabrication of C60/graphene hybrid nanostructures by physical thermal evaporation. Particularly, the proper manipulation of deposition and annealing conditions allow the control over the creation of 1D and quasi 1D C60 structures on rippled graphene. Neuroscience Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells Conrad M. Tenenbaum1, Elizabeth R. Gavis1 1Department of Molecular Biology, Princeton University Studies of neuronal morphogenesis using Drosophila larval dendritic arborization (da) neurons benefit from in situ visualization of neuronal and epidermal proteins by immunofluorescence. We describe a procedure that improves immunofluorescence analysis of da neurons and surrounding epidermal cells by removing muscle tissue from the larval body wall. Bioengineering Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix Alexandra S. Piotrowski-Daspit1, Celeste M. Nelson1,2 1Chemical and Biological Engineering, Princeton University, 2Molecular Biology, Princeton University This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates. Biology Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells Henrik D. Møller1, Rasmus K. Bojsen2, Chris Tachibana3, Lance Parsons4, David Botstein5, Birgitte Regenberg1 1Department of Biology, University of Copenhagen, 2National Veterinary Institute, Technical University of Denmark, 3Group Health Research Institute, 4Lewis-Sigler Institute for Integrative Genomics, Princeton University, 5Calico Life Sciences LLC This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation. Neuroscience Intracerebroventricular Injection of Amyloid-β Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits Hye Yun Kim1,2, Dongkeun K. Lee1, Bo-Ryehn Chung1,3, Hyunjin V. Kim1,4, YoungSoo Kim1,4 1Center for Neuro-Medicine, Korea Institute of Science and Technology, 2Research Institute, GoshenBiotech, Inc., 3Department of Chemical and Biological Engineering, Princeton University, 4Biological Chemistry Program, Korea University of Science and Technology The amyloid-β (Aβ)-injected animal model enables the administration of a defined quantity and species of Aβ fragments and reduces individual differences within each study group. This protocol describes the intracerebroventricular (ICV) injection of Aβ without stereotactic instruments, enabling the production of Alzheimer-like behavioral abnormalities in normal mice. Biology Rapid Analysis and Exploration of Fluorescence Microscopy Images Benjamin Pavie*1, Satwik Rajaram*1, Austin Ouyang2, Jason M. Altschuler1,3, Robert J. Steininger III1, Lani F. Wu1, Steven J. Altschuler1 1Green Center for Systems Biology, UT Southwestern Medical Center, 2Advanced Imaging Research Center, UT Southwestern Medical Center, 3Princeton University Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform. Biology Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters R. Scott McIsaac*1,3, Benjamin L. Oakes*1, David Botstein1,2, Marcus B. Noyes1 1The Lewis-Sigler Institute for Integrative Genomics, Princeton University, 2Department of Molecular Biology, Princeton University, 3Division of Chemistry and Chemical Engineering, California Institute of Technology This protocol describes an experimental procedure for the rapid construction of artificial transcription factors (ATFs) with cognate GFP reporters and quantification of the ATFs ability to stimulate GFP expression via flow cytometry. Immunology and Infection Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread Matthew P. Taylor1,2, Radomir Kratchmarov2, Lynn W. Enquist2 1Department of Immunology and Infectious Diseases, Montana State University, 2Department of Molecular Biology, Princeton University Live cell imaging of alphaherpes virus infections enables analysis of the dynamic events of directed transport and intercellular spread. Here, we present methodologies that utilize recombinant viral strains expressing fluorescent fusion proteins to facilitate visualization of viral assemblies during infection of primary neurons. Biology Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules James Smadbeck1, Meghan B. Peterson1, George A. Khoury1, Martin S. Taylor1, Christodoulos A. Floudas1 1Department of Chemical and Biological Engineering, Princeton University We developed computational de novo protein design methods capable of tackling several important areas of protein design. To disseminate these methods we present Protein WISDOM, an online tool for protein design (http://www.proteinwisdom.org). Starting from a structural template, design of monomeric proteins for increased stability and complexes for increased binding affinity can be performed. Biology Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae R. Scott McIsaac1,2, Sanford J. Silverman1, Lance Parsons1, Ping Xu1, Ryan Briehof1, Megan N. McClean1, David Botstein1,3 1The Lewis-Sigler Institute for Integrative Genomics, Princeton University, 2Graduate Program in Quantitative and Computational Biology, Princeton University, 3Department of Molecular Biology, Princeton University This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution. Medicine Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy Chung Wong1, Evan Vosburgh1,2, Arnold J. Levine2,3, Lei Cong2, Eugenia Y. Xu1,2 1Raymond and Beverly Sackler Foundation, 2The Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey, 3School of Natural Sciences, Institute for Advanced Study, Princeton, New Jersey We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy. Immunology and Infection Preparation of Viral DNA from Nucleocapsids Moriah L. Szpara1, Yolanda R. Tafuri1, L. W. Enquist1 1Department of Molecular Biology, Princeton University We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants. Neuroscience C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays Amanda Kauffman1, Lance Parsons2, Geneva Stein1, Airon Wills1, Rachel Kaletsky1, Coleen Murphy1 1Department of Molecular Biology, Lewis-Sigler Institute for Integrative Genomics, Princeton University, 2Lewis-Sigler Institute for Integrative Genomics, Princeton University Here we describe methods to test C. elegans associative learning and short- and long-term associative memory. These population assays employ the worms abilities to chemotax toward volatile odorants, and form positive associations upon pairing food with the chemoattractant butanone. Increasing the number of conditioning periods induces long-term memory. Neuroscience How to Create and Use Binocular Rivalry David Carmel1,2, Michael Arcaro3,4, Sabine Kastner3,4, Uri Hasson3,4 1Department of Psychology, New York University, 2Centre for Neural Science, New York University, 3Department of Psychology, Princeton University, 4Neuroscience Institute, Princeton University Binocular rivalry occurs when the eyes are presented with different images at the same location: one image dominates while the other is suppressed, and dominance alternates periodically. Rivalry is useful for investigating perceptual selection and visual awareness. Here we describe several easy methods for creating and using binocular rivalry stimuli. Biology Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap Siyuan Wang1, Hugo Arellano-Santoyo2, Peter A. Combs2, Joshua W. Shaevitz2 1Department of Molecular Biology, Lewis-Sigler Institute for Integrative Genomics, Princeton University, 2Department of Physics, Lewis-Sigler Institute for Integrative Genomics, Princeton University We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness. Biology A Rapid Technique for the Visualization of Live Immobilized Yeast Cells Karl Zawadzki1, James Broach1 1Department of Molecular Biology, Princeton University A rapid technique for the visualization of growing immobilized yeast cells, here applied to fluorescent reporters at the silent mating loci HML and HMR Biology Dissection of Larval CNS in Drosophila Melanogaster Nathaniel Hafer1, Paul Schedl1 1Department of Molecular Biology, Princeton University In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae. Biology Assay for Adhesion and Agar Invasion in S. cerevisiae Cemile G Guldal1, James Broach1 1Department of Molecular Biology, Princeton University We describe a qualitative assay for yeast adhesion and agar invasion as a measure of invasive and pseudohyphal differentiation. This simple assay can be used to assess the invasive phenotype of various mutants as well as the effects environmental cues and signaling pathways on yeast differentiation. Biology Dissection of Drosophila Ovaries Li Chin Wong1, Paul Schedl1 1Princeton University