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Labeling and Magnetic Resonance Imaging of Exosomes Isolated from Adipose Stem Cells
Alice Busato, Roberta Bonafede, Pietro Bontempi, Ilaria Scambi, Lorenzo Schiaffino, Donatella Benati, Manuela Malatesta, Andrea Sbarbati, Pasquina Marzola, Raffaella Mariotti
Adipose stem cells (ASC) represent a promising therapeutic approach for neurodegenerative diseases. Most biological effects of ASC are probably mediated by extracellular vesicles, such as exosomes, which influence the surrounding cells. Current development of exosome therapies requires efficient and
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Super‐Resolution Microscopy and Single‐Protein Tracking in Live Bacteria Using a Genetically Encoded, Photostable Fluoromodule
Saumya Saurabh, Adam M. Perez, Colin J. Comerci, Lucy Shapiro, W. E. Moerner
Visualization of dynamic protein structures in live cells is crucial for understanding the mechanisms governing biological processes. Fluorescence microscopy is a sensitive tool for this purpose. In order to image proteins in live bacteria using fluorescence microscopy, one typically genetically
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Microcontact Peeling: A Cell Micropatterning Technique for Circumventing Direct Adsorption of Proteins to Hydrophobic PDMS
Sho Yokoyama, Tsubasa S. Matsui, Shinji Deguchi
Microcontact printing (μCPr) is one of the most popular techniques used for cell micropatterning. In conventional μCPr, a polydimethylsiloxane (PDMS) stamp with microfeatures is used to adsorb extracellular matrix (ECM) proteins onto the featured surface and transfer them onto particular areas of a
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Traction Force Microscopy in 3‐Dimensional Extracellular Matrix Networks
M. Cóndor, J. Steinwachs, C. Mark, J.M. García‐Aznar, B. Fabry
Cell migration through a three‐dimensional (3‐D) matrix depends strongly on the ability of cells to generate traction forces. To overcome the steric hindrance of the matrix, cells need to generate sufficiently high traction forces but also need to distribute these forces spatially in a
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Patterning on Topography for Generation of Cell Culture Substrates with Independent Nanoscale Control of Chemical and Topographical Extracellular Matrix Cues
Emily N. Sevcik, John M. Szymanski, Quentin Jallerat, Adam W. Feinberg
The cell microenvironment plays an important role in many biological processes, including development and disease progression. Key to this is the extracellular matrix (ECM), a complex biopolymer network serving as the primary insoluble signaling network for physical, chemical, and mechanical cues.
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Labeling DNA Replication Foci to Visualize Chromosome Territories In Vivo
Apolinar Maya‐Mendoza, Dean A. Jackson
While a detailed understanding of chromatin dynamics is needed to explain how higher‐order chromatin organization influences nuclear function, the molecular principles that regulate chromatin mobility in mammalian nuclei remain largely unknown. Here we describe experimental tools to follow chromatin
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Photoactivated In Vivo Proximity Labeling
David B. Beck, Roberto Bonasio
Identification of molecular interactions is paramount to understanding how cells function. Most available technologies rely on co‐purification of a protein of interest and its binding partners. Therefore, they are limited in their ability to detect low‐affinity interactions and cannot be applied to
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Designing Drug‐Response Experiments and Quantifying their Results
Marc Hafner, Mario Niepel, Kartik Subramanian, Peter K. Sorger
We developed a Python package to help in performing drug‐response experiments at medium and high throughput and evaluating sensitivity metrics from the resulting data. In this article, we describe the steps involved in (1) generating files necessary for treating cells with the HP D300 drug
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Local Generation and Imaging of Hydrogen Peroxide in Living Cells
Yulia A. Bogdanova, Carsten Schultz, Vsevolod V. Belousov
Described here is a localized H 2 O 2 generation‐detection system consisting of a yeast D ‐amino acid oxidase (DAAO) and two spectrally distinct variants of biosensor, HyPer2 and HyPerRed based on circularly permutated yellow and red fluorescent proteins, respectively, which enables spatiotemporal
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Measuring Cancer Drug Sensitivity and Resistance in Cultured Cells
Mario Niepel, Marc Hafner, Mirra Chung, Peter K. Sorger
Measuring the potencies of small‐molecule drugs in cell lines is a critical aspect of preclinical pharmacology. Such experiments are also prototypical of high‐throughput experiments in multi‐well plates. The procedure is simple in principle, but many unrecognized factors can affect the results,
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Paper‐based Invasion Assays for Quantifying Cellular Movement in Three‐dimensional Tissue‐like Structures
C. Chad Lloyd, Matthew W. Boyce, Matthew R. Lockett
To elucidate the chemical and environmental conditions that promote invasion of cancer cells, an assay is needed in which the chemical landscape of a tumor‐like environment can be experimentally manipulated and probed. The three‐dimensional paper‐based invasion assays described here simulate poorly
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Mouse Model of Burn Wound and Infection: Thermal (Hot Air) Lesion‐Induced Immunosuppression
Henrik Calum, Niels Høiby, Claus Moser
The immunosuppression induced by thermal injury renders the burned victim susceptible to infection. A mouse model was developed to examine the immunosuppression, which was possible to induce even at a minor thermal insult of 6% total body surface area. After induction of the burn (48 hr) a
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A Fast, Easy, and Customizable Eight‐Color Flow Cytometric Method for Analysis of the Cellular Content of Bronchoalveolar Lavage Fluid in the Mouse
François Daubeuf, Julien Becker, Juan Antonio Aguilar‐Pimentel, Claudine Ebel, Martin Hrabě de Angelis, Yann Hérault, Nelly Frossard
The cell composition of bronchoalveolar lavage fluid (BAL) is an important indicator of airway inflammation. It is commonly determined by cytocentrifuging leukocytes on slides, then staining, identifying, and counting them as eosinophils, neutrophils, macrophages, or lymphocytes according to
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Monitoring Pathogen‐Induced Sickness in Mice and Rats
Daria Kolmogorova, Emma Murray, Nafissa Ismail
Sickness behavior monitoring, a technique for examining the development of sickness symptomatology following infection, is necessary in experiments studying neurochemical and physiological changes associated with pathogen‐induced immune activation. However, the results of sickness behavior
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Application of SWATH Proteomics to Mouse Biology
Yibo Wu, Evan G. Williams, Ruedi Aebersold
The quantitative measurement of the proteome has been shown to yield new insights into physiology and cell biology that cannot be determined from the genome and transcriptome because the quantitative relationship between transcriptome and proteome is complex. MS‐based proteomics techniques, such as
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Microbiota Analysis Using an Illumina MiSeq Platform to Sequence 16S rRNA Genes
Alexis Rapin, Céline Pattaroni, Benjamin J. Marsland, Nicola L. Harris
The microbiota have been shown to play an important role in diverse biological processes including immunity, metabolism, and digestion. Assessing the exact composition of the microbiota has proven challenging due to the often unknown growth specificities of its members, and culture‐based approaches
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Nucleobase Protection of Deoxyribo‐ and Ribonucleosides
Geeta Meher, Nabin K. Meher, Radhakrishnan P. Iyer
Oligonucleotides carrying a variety of chemical modifications including conjugates are finding increasing applications in therapeutics, diagnostics, functional genomics, proteomics, and as research tools in chemical and molecular biology. The successful synthesis of oligonucleotides primarily
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Synthesis of Oligodeoxynucleotides Containing a C8‐2′‐Deoxyguanosine Adduct Formed by the Carcinogen 3‐Nitrobenzanthrone
Arindom Chatterjee, Chanchal K. Malik, Ashis K. Basu
This unit describes the detailed procedure in five parts for the synthesis of the C8‐2′‐deoxyguanosine‐3‐aminobenzanthrone adduct located in a desired site in an oligonucleotide. The synthesis of the protected 2′‐deoxyguanosine, O 6 ‐benzyl‐ N 2 ‐DMTr‐3′‐5′‐bisTBDMS‐C8‐Br‐2′‐deoxyguanosine, is
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Carbocyclic C‐C Bond Formation: Intramolecular Radical Ring Closure to Yield Diastereomerically Pure (7′S‐Me‐ or 7′R‐Me‐) Carba‐LNA Nucleotide Analogs
Oleksandr Plashkevych, Ram Shankar Upadhayaya, Jyoti Chattopadhyaya
In light of the impressive gene‐silencing properties of carba‐LNA modified oligo DNA and RNA, both in antisense RNA and siRNA approaches, which have been confirmed as proof‐of‐concept for biochemical applications in post‐transcriptional gene silencing, we envision the true potential of carba‐LNA
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Engineered Polymerases with Altered Substrate Specificity: Expression and Purification
Ali Nikoomanzar, Matthew R. Dunn, John C. Chaput
Polymerase engineering is making it possible to synthesize xeno‐nucleic acid polymers (XNAs) with diverse backbone structures and chemical functionality. The ability to copy genetic information back and forth between DNA and XNA has led to a new field of science known as synthetic genetics, which
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A High‐Throughput Process for the Solid‐Phase Purification of Synthetic DNA Sequences
Andrzej Grajkowski, Jacek Cieślak, Serge L. Beaucage
An efficient process for the purification of synthetic phosphorothioate and native DNA sequences is presented. The process is based on the use of an aminopropylated silica gel support functionalized with aminooxyalkyl functions to enable capture of DNA sequences through an oximation reaction with
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Water‐Medium Synthesis of Nucleoside 5′‐Polyphosphates
Anaïs Depaix, Suzanne Peyrottes, Béatrice Roy
This unit describes a one‐pot, two step synthesis of ribonucleoside 5′‐di‐ and 5′‐triphosphates, as well as their purification. The first step of the synthesis involves the activation of an unprotected ribonucleoside 5′‐monophosphate with 2‐chloro‐1,3‐dimethylimidazolinium hexafluorophosphate and
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Stereoselective Synthesis of 4′‐Selenonucleosides via the Seleno‐Michael Reaction
Pramod K. Sahu, Dnyandev B. Jarhad, Gyudong Kim, Lak Shin Jeong
5′‐Homo‐4′‐selenonucleosides, a class of next‐generation nucleosides, are synthesized from D ‐ribose via a 4‐selenosugar intermediate. The key step in synthesizing this intermediate is a seleno‐Michael reaction. 5′‐Homo‐4′‐selenouridine and ‐adenosine are prepared using Pummerer‐type and Vorbrüggen
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Quantification of Grass Colonization by Associative Bacteria
Eduardo Balsanelli, Fábio de Oliveira Pedrosa, Emanuel Maltempi de Souza
There is a growing interest in the use of plant growth–promoting rhizobacteria to improve crop productivity as a partial substitute for nitrogen fertilizer. Bacteria‐colonizing plants may be epiphytic or endophytic. This article describes reproducible protocols to quantify the level of colonization
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Plant Microbiome Identification and Characterization
Sarah L. Lebeis
To fully exploit the potential of plant microbiome alterations to improve plant health, reliable methods must be used to prepare and characterize microbiome samples. The power of culture‐independent studies is that they allow the characterization of novel microbial community members, but only
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Generation of Maize (Zea mays) Doubled Haploids via Traditional Methods
Kimberly Vanous, Adam Vanous, Ursula K. Frei, Thomas Lübberstedt
Commercial maize hybrid production has corroborated the usefulness of producing inbred lines; however, the delivery of new lines has always been a major time constraint in breeding programs. Traditional methods for developing inbred lines typically require 6 to 10 generations of self‐pollination to
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Handling Fast‐Flowering Mini‐Maize
Morgan E. McCaw, James A. Birchler
Maize ( Zea mays ) has a long history as a model organism for genetic analysis due to its ease of crossing, relatively large number of progeny, and ability to determine many genotypic traits through phenotypic kernel markers. Fast‐Flowering Mini‐Maize A and B (FFMM) are two recently developed lines
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Plant iTRAQ‐Based Proteomics
Pubudu P. Handakumbura, Kim K. Hixson, Samuel O. Purvine, Christer Jansson, Ljiljana Paša‐Tolić
We present a simple one‐pot extraction protocol, which rapidly isolates hydrophilic metabolites, lipids, and proteins from the same pulverized plant sample. Also detailed is a global plant proteomics sample preparation method utilizing iTRAQ multiplexing reagents that enables deep proteome coverage
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The Ussing Chamber Assay to Study Drug Metabolism and Transport in the Human Intestine
Beatrice Kisser, Eva Mangelsen, Caroline Wingolf, Lars Ivo Partecke, Claus‐Dieter Heidecke, Christer Tannergren, Stefan Oswald, Markus Keiser
The Ussing chamber is an old but still powerful technique originally designed to study the vectorial transport of ions through frog skin. This technique is also used to investigate the transport of chemical agents through the intestinal barrier as well as drug metabolism in enterocytes, both of
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Label‐Free Dynamic Mass Redistribution and Bio‐Impedance Methods for Drug Discovery
Manuel Grundmann
Label‐free biosensors are increasingly employed in drug discovery. Cell‐based biosensors provide valuable insights into the biological consequences of exposing cells and tissues to chemical agents and the underlying molecular mechanisms associated with these effects. Optical biosensors based on the
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Overview of Transgenic Mouse Models of Myeloproliferative Neoplasms (MPNs)
Andrew Dunbar, Abbas Nazir, Ross Levine
Myeloproliferative neoplasms (MPNs) are a class of hematologic diseases characterized by aberrant proliferation of one or more myeloid lineages and progressive bone marrow fibrosis. In 2005, seminal work by multiple groups identified the JAK2V617F mutation in a significant fraction of MPN patients.
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