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36 ARTICLES PUBLISHED IN JoVE

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Biology

Dissection of Drosophila Ovaries
Li Chin Wong 1, Paul Schedl 1
1Princeton University

Dissection of Drosophila Ovaries

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Biology

Assay for Adhesion and Agar Invasion in S. cerevisiae
Cemile G Guldal 1, James Broach 1
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.

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Biology

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells
Karl Zawadzki 1, James Broach 1
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

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Biology

Dissection of Larval CNS in Drosophila Melanogaster
Nathaniel Hafer 1, Paul Schedl 1
1Department of Molecular Biology, Princeton University

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

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Biology

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap
Siyuan Wang 1, Hugo Arellano-Santoyo 2, Peter A. Combs 2, Joshua W. Shaevitz 2
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.

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Neuroscience

How to Create and Use Binocular Rivalry
David Carmel 1,2, Michael Arcaro 3,4, Sabine Kastner 3,4, Uri Hasson 3,4
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.

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Neuroscience

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays
Amanda Kauffman 1, Lance Parsons 2, Geneva Stein 1, Airon Wills 1, Rachel Kaletsky 1, Coleen Murphy 1
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.

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Immunology and Infection

Preparation of Viral DNA from Nucleocapsids
Moriah L. Szpara 1, Yolanda R. Tafuri 1, L. W. Enquist 1
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.

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Biology

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR
Xiuchun Ge 1, Ping Xu 1
1The Philips Institute of Oral and Craniofacial Molecular Biology, Virginia Commonwealth University

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

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Biology

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae
R. Scott McIsaac 1,2, Sanford J. Silverman 1, Lance Parsons 1, Ping Xu 1, Ryan Briehof 1, Megan N. McClean 1, David Botstein 1,3
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.

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Immunology and Infection

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread
Matthew P. Taylor 1,2, Radomir Kratchmarov 2, Lynn W. Enquist 2
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.

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JoVE Journal

Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters
R. Scott McIsaac *1,3, Benjamin L. Oakes *1, David Botstein 1,2, Marcus B. Noyes 1
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.

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Biology

Rapid Analysis and Exploration of Fluorescence Microscopy Images
Benjamin Pavie *1, Satwik Rajaram *1, Austin Ouyang 2, Jason M. Altschuler 1,3, Robert J. Steininger III 1, Lani F. Wu 1, Steven J. Altschuler 1
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.

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Medicine

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging
Mikhail Paveliev 1, Mikhail Kislin 1, Dmitry Molotkov 1, Mikhail Yuryev 1, Heikki Rauvala 1, Leonard Khiroug 1
1Neuroscience Center, University of Helsinki

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

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Behavior

Flat-floored Air-lifted Platform: A New Method for Combining Behavior with Microscopy or Electrophysiology on Awake Freely Moving Rodents
Mikhail Kislin 1, Ekaterina Mugantseva 1, Dmitry Molotkov 1, Natalia Kulesskaya 1, Stanislav Khirug 1, Ilya Kirilkin 2, Evgeny Pryazhnikov 1,2, Julia Kolikova 2, Dmytro Toptunov 2, Mikhail Yuryev 1, Rashid Giniatullin 3, Vootele Voikar 4, Claudio Rivera 1, Heikki Rauvala 1, Leonard Khiroug 1
1Neuroscience Center, University of Helsinki, 2Neurotar LTD, 3A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, 4Laboratory Animal Center, University of Helsinki

This method creates a tangible, familiar environment for the mouse to navigate and explore during microscopic imaging or single-cell electrophysiological recordings, which require firm fixation of the animal’s head.

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Developmental Biology

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line
Mackenzie M. Shipley 1, Colleen A. Mangold 1, Moriah L. Szpara 1
1Department of Biochemistry and Molecular Biology, The Huck Institutes of the Life Sciences, The Pennsylvania State University

It is critical in neurobiology and neurovirology to have a reliable, replicable in vitro system that serves as a translational model for what occurs in vivo in human neurons. This protocol describes how to culture and differentiate SH-SY5Y human neuroblastoma cells into viable neurons for use in in vitro applications.

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Neuroscience

Intracerebroventricular Injection of Amyloid-β Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits
Hye Yun Kim 1,2, Dongkeun K. Lee 1, Bo-Ryehn Chung 1,3, Hyunjin V. Kim 1,4, YoungSoo Kim 1,4
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.

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Biology

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells
Henrik D. Møller 1, Rasmus K. Bojsen 2, Chris Tachibana 3, Lance Parsons 4, David Botstein 5, Birgitte Regenberg 1
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.

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Bioengineering

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix
Alexandra S. Piotrowski-Daspit 1, Celeste M. Nelson 1,2
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.

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Neuroscience

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells
Conrad M. Tenenbaum 1, Elizabeth R. Gavis 1
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.

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Bioengineering

Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications
Cameron J. Stewart 1, Megan N. McClean 1
1Department of Biomedical Engineering, University of Wisconsin-Madison

We designed a continuous culturing apparatus for use with optogenetic systems to illuminate cultures of microbes and regularly image cells in the effluent with an inverted microscope. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses to illumination can be measured over several days.

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Chemistry

Preparation and Characterization of C60/Graphene Hybrid Nanostructures
Chuanhui Chen 1, Adam Mills 1,2, Husong Zheng 1, Yanlong Li 1, Chenggang Tao 1
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.

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Bioengineering

Generation of Heterogeneous Drug Gradients Across Cancer Populations on a Microfluidic Evolution Accelerator for Real-Time Observation
Ke-Chih Lin 1, Gonzalo Torga 2, Yusha Sun 1, Kenneth J. Pienta 2, James C. Sturm 1, Robert H. Austin 1
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.

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Immunology and Infection

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization
Benjamin P. Bratton 2, Brody Barton 1, Randy M. Morgenstein 1
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.

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Bioengineering

Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior
Francesco Carrara 1, Douglas R. Brumley 2, Andrew M. Hein 3, Yutaka Yawata 4,5, M. Mehdi Salek 1, Kang Soo Lee 1, Elzbieta Sliwerska 1, Simon A. Levin 6, Roman Stocker 1
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.

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Immunology and Infection

Mouse Footpad Inoculation Model to Study Viral-Induced Neuroinflammatory Responses
Kathlyn Laval 1, Carola J. Maturana 2, Lynn W. Enquist 1
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.

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Cancer Research

A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth
David Jelinek 1,2, Ellen Ran Zhang 1,2,3, Aaron Ambrus 1,2, Erin Haley 3, Emily Guinn 1,2, Austin Vo 1,2, Peter Le 1,2, Ayse Elif Kesaf 1,2, Jennifer Nguyen 1,2, Lily Guo 1,2, Destiny Frederick 1,2, Zhengyang Sun 1,2, Natalie Guo 3, Parker Sevier 1,2, Eric Bilotta 1,2, Kaiser Atai 1,2,4, Laurent Voisin 1,2, Hilary A. Coller 1,2,4
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.

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Biology

Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging
Kelly Veerasammy *1,2, Yuki X. Chen *1,2, Sami Sauma *1, Mathilde Pruvost 1, David K. Dansu 1, Tenzin Choetso 1,2, Tiffany Zhong 3, Damien Marechal 1, Patrizia Casaccia 1, Rinat Abzalimov 4,5, Ye He 1,5
1The Graduate Center - Advanced Science Research Center, Neuroscience Initiative, The City University of New York, 2The City College of New York, CUNY, 3The Bronx High School of Science, 4The Graduate Center - Advanced Science Research Center, Structural Biology Initiative, The City University of New York, 5The Graduate Center - Advanced Science Research Center, MALDI MS Imaging Joint Core Facility, The City University of New York

The goal of this protocol is to provide detailed guidance on the sample preparation when planning for experiments using MALDI MSI to maximize metabolic and molecular detection in biological samples.

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Biology

Profiling of H3K4me3 Modification in Plants using Cleavage under Targets and Tagmentation
Xiaoyuan Tao *1,2, Mengtao Gao *3, Siyuan Wang *1, Xueying Guan 1,4
1College of Agriculture and Biotechnology, Zhejiang University, 2Central Laboratory, State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Zhejiang Academy of Agricultural Sciences, 3State Key Laboratory of Crop Genetics and Germplasm Enhancement, Cotton Hybrid R & D Engineering Center (the Ministry of Education), College of Agriculture, Nanjing Agricultural University, 4Hainan Institute of Zhejiang University

Cleavage under targets and tagmentation (CUT&Tag) is an efficient chromatin epigenomic profiling strategy. This protocol presents a refined CUT&Tag strategy for the profiling of histone modifications in plants.

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Neuroscience

A Flexible Platform for Monitoring Cerebellum-Dependent Sensory Associative Learning
Gerard Joey Broussard 1, Mikhail Kislin 1, Caroline Jung 1, Samuel S. -H. Wang 1
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.

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Bioengineering

Light-Controlled Fermentations for Microbial Chemical and Protein Production
Shannon M. Hoffman *1, Makoto A. Lalwani *1, José L. Avalos 1,2,3,4
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.

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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 Choetso 1,4, Tiffany Zhong 7, Muniyat A. Choudhury 1,4,5, Cory Weng 1,4,5, Ethan Xu 8, Mayan A. Hein 9,10, Rinat Abzalimov 2, Ye He 1
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.

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Developmental Biology

Optogenetic Inhibition of Rho1-Mediated Actomyosin Contractility Coupled with Measurement of Epithelial Tension in Drosophila Embryos
Hanqing Guo 1,2, Michael Swan 3, Bing He 1
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.

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Bioengineering

3D Printing Bacteria to Study Motility and Growth in Complex 3D Porous Media
R. Kōnane Bay 1,2, Anna M. Hancock 2, Arabella S. Dill-Macky 2, Hao Nghi Luu 2, Sujit S. Datta 2
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.

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Bioengineering

Synthesizing Lipid Nanoparticles by Turbulent Flow in Confined Impinging Jet Mixers
Sai Nikhil Subraveti 1, Brian K. Wilson 1, Navid Bizmark 1, Jason Liu 1, Robert K. Prud'homme 1
1Department of Chemical and Biological Engineering, Princeton University

A detailed protocol for synthesizing lipid nanoparticles (LNPs) using confined impinging jet (CIJ) mixer technologies, including a two-jet CIJ and a four-jet multi-inlet vortex mixer (µMIVM), is demonstrated. The CIJ mixers generate reproducible, turbulent micro-mixing environments, resulting in the production of monodisperse LNPs.

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