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Nature Protocols

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https://www.readbyqxmd.com/read/28079880/nonradioactive-quantification-of-autophagic-protein-degradation-with-l-azidohomoalanine-labeling
#1
Jigang Wang, Jianbin Zhang, Yew Mun Lee, Shukie Ng, Yin Shi, Zi-Chun Hua, Qingsong Lin, Han-Ming Shen
At present, several assays that use radioisotope labeling to quantify the degradation of long-lived proteins have been developed to measure autophagic flux. Here, we describe a nonradioactive pulse-chase protocol using L-azidohomoalanine (AHA) labeling to quantify long-lived protein degradation during autophagy. AHA is used as a surrogate for L-methionine, and, when added to cultured cells grown in methionine-free medium, AHA is incorporated into proteins during de novo protein synthesis. After a chase period to remove short-lived proteins, autophagy is induced by starvation or other stimuli...
December 2017: Nature Protocols
https://www.readbyqxmd.com/read/28726849/o2-controllable-hydrogels-for-studying-cellular-responses-to-hypoxic-gradients-in-three-dimensions-in-vitro-and-in-vivo
#2
Daniel M Lewis, Michael R Blatchley, Kyung Min Park, Sharon Gerecht
Oxygen (O2) acts as a potent upstream regulator of cell function. In both physiological and pathophysiological microenvironments, the O2 concentration is not uniformly distributed but instead follows a gradient that depends on distance from oxygen-carrying blood vessels. Such gradients have a particularly important role in development, tissue regeneration, and tumor growth. In this protocol, we describe how to use our previously reported gelatin-based O2-controllable hydrogels that can provide hypoxic microenvironments in vitro...
August 2017: Nature Protocols
https://www.readbyqxmd.com/read/28726848/patch-clamp-technique-to-characterize-ion-channels-in-enlarged-individual-endolysosomes
#3
Cheng-Chang Chen, Chunlei Cang, Stefanie Fenske, Elisabeth Butz, Yu-Kai Chao, Martin Biel, Dejian Ren, Christian Wahl-Schott, Christian Grimm
According to proteomics analyses, more than 70 different ion channels and transporters are harbored in membranes of intracellular compartments such as endosomes and lysosomes. Malfunctioning of these channels has been implicated in human diseases such as lysosomal storage disorders, neurodegenerative diseases and metabolic pathologies, as well as in the progression of certain infectious diseases. As a consequence, these channels have engendered very high interest as future drug targets. Detailed electrophysiological characterization of intracellular ion channels is lacking, mainly because standard methods to analyze plasma membrane ion channels, such as the patch-clamp technique, are not readily applicable to intracellular organelles...
August 2017: Nature Protocols
https://www.readbyqxmd.com/read/28726847/mapping-genome-wide-transcription-factor-binding-sites-using-dap-seq
#4
Anna Bartlett, Ronan C O'Malley, Shao-Shan Carol Huang, Mary Galli, Joseph R Nery, Andrea Gallavotti, Joseph R Ecker
To enable low-cost, high-throughput generation of cistrome and epicistrome maps for any organism, we developed DNA affinity purification sequencing (DAP-seq), a transcription factor (TF)-binding site (TFBS) discovery assay that couples affinity-purified TFs with next-generation sequencing of a genomic DNA library. The method is fast, inexpensive, and more easily scaled than chromatin immunoprecipitation sequencing (ChIP-seq). DNA libraries are constructed using native genomic DNA from any source of interest, preserving cell- and tissue-specific chemical modifications that are known to affect TF binding (such as DNA methylation) and providing increased specificity as compared with in silico predictions based on motifs from methods such as protein-binding microarrays (PBMs) and systematic evolution of ligands by exponential enrichment (SELEX)...
August 2017: Nature Protocols
https://www.readbyqxmd.com/read/28703790/a-fluorescence-based-imaging-method-to-measure-in-vitro-and-in-vivo-mitophagy-using-mt-keima
#5
Nuo Sun, Daniela Malide, Jie Liu, Ilsa I Rovira, Christian A Combs, Toren Finkel
Mitophagy is a cellular process that selectively removes damaged, old or dysfunctional mitochondria. Defective mitophagy is thought to contribute to normal aging and to various neurodegenerative and cardiovascular diseases. Previous methods used to detect mitophagy in vivo were cumbersome, insensitive and difficult to quantify. We created a transgenic mouse model that expresses the pH-dependent fluorescent protein mt-Keima in order to more readily assess mitophagy. Keima is a pH-sensitive, dual-excitation ratiometric fluorescent protein that also exhibits resistance to lysosomal proteases...
August 2017: Nature Protocols
https://www.readbyqxmd.com/read/28703789/capturing-suboptical-dynamic-structures-in-lipid-bilayer-patches-formed-from-free-standing-giant-unilamellar-vesicles
#6
Tripta Bhatia, Flemming Cornelius, John H Ipsen
There is accumulating evidence that the small-scale lateral organization of biological membranes has a crucial role in signaling and trafficking in cells. However, it has been difficult to characterize these features with existing methods for preparing and analyzing freestanding membranes, because the dynamics occurs below the optical resolution possible with these protocols. We have developed a protocol that permits the imaging of lipid nanodomains and lateral protein organization in membranes of giant unilamellar vesicles (GUVs)...
August 2017: Nature Protocols
https://www.readbyqxmd.com/read/28703788/preparation-of-a-trp-bodipy-fluorogenic-amino-acid-to-label-peptides-for-enhanced-live-cell-fluorescence-imaging
#7
Lorena Mendive-Tapia, Ramon Subiros-Funosas, Can Zhao, Fernando Albericio, Nick D Read, Rodolfo Lavilla, Marc Vendrell
Fluorescent peptides are valuable tools for live-cell imaging because of the high specificity of peptide sequences for their biomolecular targets. When preparing fluorescent versions of peptides, labels must be introduced at appropriate positions in the sequences to provide suitable reporters while avoiding any impairment of the molecular recognition properties of the peptides. This protocol describes the preparation of the tryptophan (Trp)-based fluorogenic amino acid Fmoc-Trp(C2-BODIPY)-OH and its incorporation into peptides for live-cell fluorescence imaging-an approach that is applicable to most peptide sequences...
August 2017: Nature Protocols
https://www.readbyqxmd.com/read/28683064/murine-chronic-lymph-node-window-for-longitudinal-intravital-lymph-node-imaging
#8
Eelco F J Meijer, Han-Sin Jeong, Ethel R Pereira, Thomas A Ruggieri, Cedric Blatter, Benjamin J Vakoc, Timothy P Padera
Chronic imaging windows in mice have been developed to allow intravital microscopy of many different organs and have proven to be of paramount importance in advancing our knowledge of normal and disease processes. A model system that allows long-term intravital imaging of lymph nodes would facilitate the study of cell behavior in lymph nodes during the generation of immune responses in a variety of disease settings and during the formation of metastatic lesions in cancer-bearing mice. We describe a chronic lymph node window (CLNW) surgical preparation that allows intravital imaging of the inguinal lymph node in mice...
August 2017: Nature Protocols
https://www.readbyqxmd.com/read/28683063/shear-thinning-and-self-healing-hydrogels-as-injectable-therapeutics-and-for-3d-printing
#9
Claudia Loebel, Christopher B Rodell, Minna H Chen, Jason A Burdick
The design of injectable hydrogel systems addresses the growing demand for minimally invasive approaches for local and sustained delivery of therapeutics. We developed a class of hyaluronic acid (HA) hydrogels that form through noncovalent guest-host interactions, undergo disassembly (shear-thinning) when injected through a syringe and then reassemble within seconds (self-healing) when shear forces are removed. Its unique properties enable the use of this hydrogel system for numerous applications, such as injection in vivo (including with cells and therapeutic molecules) or as a 'bioink' in 3D-printing applications...
August 2017: Nature Protocols
https://www.readbyqxmd.com/read/28683062/use-of-luciferase-probes-to-measure-atp-in-living-cells-and-animals
#10
Giampaolo Morciano, Alba Clara Sarti, Saverio Marchi, Sonia Missiroli, Simonetta Falzoni, Lizzia Raffaghello, Vito Pistoia, Carlotta Giorgi, Francesco Di Virgilio, Paolo Pinton
ATP, the energy exchange factor that connects anabolism and catabolism, is required for major reactions and processes that occur in living cells, such as muscle contraction, phosphorylation and active transport. ATP is also the key molecule in extracellular purinergic signaling mechanisms, with an established crucial role in inflammation and several additional disease conditions. Here, we describe detailed protocols to measure the ATP concentration in isolated living cells and animals using luminescence techniques based on targeted luciferase probes...
August 2017: Nature Protocols
https://www.readbyqxmd.com/read/28686587/qmotor-a-set-of-rules-for-sensitive-robust-and-quantitative-measurement-of-motor-performance-in-mice
#11
Laura M Luh, Indrajit Das, Anne Bertolotti
Phenotypic analysis of mouse models of human diseases is essential to understanding the underlying disease mechanisms and to developing therapeutics. Many models of neurodegenerative diseases are associated with motor dysfunction, a powerful readout for the disease. We describe here a set of measures to quantitatively monitor early disease onset and progression. We named this set of rules qMotor because it enables sensitive, robust and quantitative measurement of motor performance in 3 d. qMotor can be used to assess early disease onset, before paralysis, as well as disease progression in diverse mouse models, and can be exploited to define robust and humane experimental end points, thereby reducing animal suffering...
July 2017: Nature Protocols
https://www.readbyqxmd.com/read/28686586/quantifying-transcription-factor-dna-binding-in-single-cells-in-vivo-with-photoactivatable-fluorescence-correlation-spectroscopy
#12
Ziqing Winston Zhao, Melanie D White, Yanina D Alvarez, Jennifer Zenker, Stephanie Bissiere, Nicolas Plachta
Probing transcription factor (TF)-DNA interactions remains challenging in complex in vivo systems such as mammalian embryos, especially when TF copy numbers and fluorescence background are high. To address this difficulty, fluorescence correlation spectroscopy (FCS) can be combined with the use of photoactivatable fluorescent proteins to achieve selective photoactivation of a subset of tagged TF molecules. This approach, termed paFCS, enables FCS measurements within single cell nuclei inside live embryos, and obtains autocorrelation data of a quality previously only attainable in simpler in vitro cell culture systems...
July 2017: Nature Protocols
https://www.readbyqxmd.com/read/28686585/constructing-cellular-niche-properties-by-localized-presentation-of-wnt-proteins-on-synthetic-surfaces
#13
Molly Lowndes, Sergi Junyent, Shukry J Habib
Wnt signaling is crucial during embryonic development and for the maintenance of adult tissues. Depending on the tissue type, the Wnt pathway can promote stem cell self-renewal and/or direct lineage commitment. Wnt proteins are subject to lipid modification, often restricting them to act in a localized manner on responsive cells. Most methods for inducing Wnt signaling in stem cell cultures do not control the spatial presentation of the protein. To recreate the local presentation of Wnt proteins often seen in vivo, we previously developed a method to immobilize the protein onto synthetic surfaces...
July 2017: Nature Protocols
https://www.readbyqxmd.com/read/28686584/assessing-recent-and-remote-associative-olfactory-memory-in-rats-using-the-social-transmission-of-food-preference-paradigm
#14
Benjamin Bessières, Olivier Nicole, Bruno Bontempi
Rats have the ability to learn about potential food sources by sampling their odors on the breath of conspecifics. Although this ethologically based social behavior has been transposed to the laboratory to probe nonspatial associative olfactory memory, only a few studies have taken full advantage of its unique features to examine the organization of recently and remotely acquired information. We provide a set of standardized procedures and technical refinements that are particularly useful in achieving this goal while minimizing confounding factors...
July 2017: Nature Protocols
https://www.readbyqxmd.com/read/28686583/interfacing-3d-magnetic-twisting-cytometry-with-confocal-fluorescence-microscopy-to-image-force-responses-in-living-cells
#15
Yuejin Zhang, Fuxiang Wei, Yeh-Chuin Poh, Qiong Jia, Junjian Chen, Junwei Chen, Junyu Luo, Wenting Yao, Wenwen Zhou, Wei Huang, Fang Yang, Yao Zhang, Ning Wang
Cells and tissues can undergo a variety of biological and structural changes in response to mechanical forces. Only a few existing techniques are available for quantification of structural changes at high resolution in response to forces applied along different directions. 3D-magnetic twisting cytometry (3D-MTC) is a technique for applying local mechanical stresses to living cells. Here we describe a protocol for interfacing 3D-MTC with confocal fluorescence microscopy. In 3D-MTC, ferromagnetic beads are bound to the cell surface via surface receptors, followed by their magnetization in any desired direction...
July 2017: Nature Protocols
https://www.readbyqxmd.com/read/28686582/measurement-of-drug-target-engagement-in-live-cells-by-two-photon-fluorescence-anisotropy-imaging
#16
Claudio Vinegoni, Paolo Fumene Feruglio, Christian Brand, Sungon Lee, Antoinette E Nibbs, Shawn Stapleton, Sunil Shah, Ignacy Gryczynski, Thomas Reiner, Ralph Mazitschek, Ralph Weissleder
The ability to directly image and quantify drug-target engagement and drug distribution with subcellular resolution in live cells and whole organisms is a prerequisite to establishing accurate models of the kinetics and dynamics of drug action. Such methods would thus have far-reaching applications in drug development and molecular pharmacology. We recently presented one such technique based on fluorescence anisotropy, a spectroscopic method based on polarization light analysis and capable of measuring the binding interaction between molecules...
July 2017: Nature Protocols
https://www.readbyqxmd.com/read/28686581/cancer-imaging-using-surface-enhanced-resonance-raman-scattering-nanoparticles
#17
Stefan Harmsen, Matthew A Wall, Ruimin Huang, Moritz F Kircher
The unique spectral signatures and biologically inert compositions of surface-enhanced resonance Raman scattering (SERRS) nanoparticles make them promising contrast agents for in vivo cancer imaging. Our SERRS nanoparticles consist of a 60-nm gold nanoparticle core that is encapsulated in a 15-nm-thick silica shell wherein the resonant Raman reporter is embedded. Subtle aspects of their preparation can shift their limit of detection by orders of magnitude. In this protocol, we present the optimized, step-by-step procedure for generating reproducible SERRS nanoparticles with femtomolar (10(-15) M) limits of detection...
July 2017: Nature Protocols
https://www.readbyqxmd.com/read/28617451/biological-and-chemical-strategies-for-exploring-inter-and-intra-kingdom-communication-mediated-via-bacterial-volatile-signals
#18
Mohamed A Farag, Geun Cheol Song, Yong-Soon Park, Bianca Audrain, Soohyun Lee, Jean-Marc Ghigo, Joseph W Kloepper, Choong-Min Ryu
Airborne chemical signals emitted by bacteria influence the behavior of other bacteria and plants. We present an overview of in vitro methods for evaluating bacterial and plant responses to bacterial volatile compounds (BVCs). Three types of equipment have been used to physically separate the bacterial test strains from either other bacterial strains or plants (in our laboratory we use either Arabidopsis or tobacco plant seedlings): a Petri dish containing two compartments (BI Petri dish); two Petri dishes connected with tubing; and a microtiter-based assay...
July 2017: Nature Protocols
https://www.readbyqxmd.com/read/28617450/a-human-intestinal-m-cell-like-model-for-investigating-particle-antigen-and-microorganism-translocation
#19
Ana Beloqui, David J Brayden, Per Artursson, Véronique Préat, Anne des Rieux
The specialized microfold cells (M cells) in the follicle-associated epithelium (FAE) of intestinal Peyer's patches serve as antigen-sampling cells of the intestinal innate immune system. Unlike 'classical' enterocytes, they are able to translocate diverse particulates without digesting them. They act as pathways for microorganism invasion and mediate food tolerance by transcellular transport of intestinal microbiota and antigens. Their ability to transcytose intact particles can be used to develop oral drug delivery and oral immunization strategies...
July 2017: Nature Protocols
https://www.readbyqxmd.com/read/28617449/assessment-of-social-transmission-of-threats-in-humans-using-observational-fear-conditioning
#20
Jan Haaker, Armita Golkar, Ida Selbing, Andreas Olsson
Across the human life span, fear is often acquired indirectly by observation of the emotional expressions of others. The observational fear conditioning protocol was previously developed as a laboratory model for investigating socially acquired threat responses. This protocol serves as a suitable alternative to the widely used Pavlovian fear conditioning, in which threat responses are acquired through direct experiences. In the observational fear conditioning protocol, the participant (observer) watches a demonstrator being presented with a conditioned stimulus (CS) paired with an aversive unconditioned stimulus (US)...
July 2017: Nature Protocols
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