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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/28426026/generation-of-multipotent-induced-cardiac-progenitor-cells-from-mouse-fibroblasts-and-potency-testing-in-ex-vivo-mouse-embryos
#2
Pratik A Lalit, Adriana M Rodriguez, Karen M Downs, Timothy J Kamp
Here we describe a protocol to generate expandable and multipotent induced cardiac progenitor cells (iCPCs) from mouse adult fibroblasts using forced expression of Mesp1, Tbx5, Gata4, Nkx2.5 and Baf60c (MTGNB) along with activation of Wnt and JAK/STAT signaling. This method does not use iPS cell factors and thus differs from cell activation and signaling-directed (CASD) reprogramming to cardiac progenitors. Our method is specific to direct CPC reprogramming, whereas CASD reprogramming can generate various cell types depending on culture conditions and raises the possibility of transitioning through a pluripotent cell state...
May 2017: Nature Protocols
https://www.readbyqxmd.com/read/28426025/modular-low-light-microscope-for-imaging-cellular-bioluminescence-and-radioluminescence
#3
Tae Jin Kim, Silvan Türkcan, Guillem Pratx
Low-light microscopy methods are receiving increased attention as new applications have emerged. One such application is to allow longitudinal imaging of light-sensitive cells with no phototoxicity and no photobleaching of fluorescent biomarkers. Another application is for imaging signals that are inherently dim and undetectable using standard microscopy techniques, such as bioluminescence, chemiluminescence or radioluminescence. In this protocol, we provide instructions on how to build a modular low-light microscope (1-4 d) by coupling two microscope objective lenses, back to back from each other, using standard optomechanical components...
May 2017: Nature Protocols
https://www.readbyqxmd.com/read/28406496/strategic-and-practical-guidelines-for-successful-structured-illumination-microscopy
#4
Justin Demmerle, Cassandravictoria Innocent, Alison J North, Graeme Ball, Marcel Müller, Ezequiel Miron, Atsushi Matsuda, Ian M Dobbie, Yolanda Markaki, Lothar Schermelleh
Linear 2D- or 3D-structured illumination microscopy (SIM or3D-SIM, respectively) enables multicolor volumetric imaging of fixed and live specimens with subdiffraction resolution in all spatial dimensions. However, the reliance of SIM on algorithmic post-processing renders it particularly sensitive to artifacts that may reduce resolution, compromise data and its interpretations, and drain resources in terms of money and time spent. Here we present a protocol that allows users to generate high-quality SIM data while accounting and correcting for common artifacts...
May 2017: Nature Protocols
https://www.readbyqxmd.com/read/28406495/quantitative-3d-structured-illumination-microscopy-of-nuclear-structures
#5
Felix Kraus, Ezequiel Miron, Justin Demmerle, Tsotne Chitiashvili, Alexei Budco, Quentin Alle, Atsushi Matsuda, Heinrich Leonhardt, Lothar Schermelleh, Yolanda Markaki
3D structured illumination microscopy (3D-SIM) is the super-resolution technique of choice for multicolor volumetric imaging. Here we provide a validated sample preparation protocol for labeling nuclei of cultured mammalian cells, image acquisition and registration practices, and downstream image analysis of nuclear structures and epigenetic marks. Using immunostaining and replication labeling combined with image segmentation, centroid mapping and nearest-neighbor analyses in open-source environments, 3D maps of nuclear structures are analyzed in individual cells and normalized to fluorescence standards on the nanometer scale...
May 2017: Nature Protocols
https://www.readbyqxmd.com/read/28384139/antifungal-drug-testing-by-combining-minimal-inhibitory-concentration-testing-with-target-identification-by-gas-chromatography-mass-spectrometry
#6
Christoph Müller, Ulrike Binder, Franz Bracher, Martin Giera
Fungal infections and their increasing resistance to antibiotics are an emerging threat to public health. Novel antifungal drugs, as well technologies that can help us bolster the antimicrobial pipeline and understand resistance mechanisms, are needed. The ergosterol biosynthetic pathway is one potential target for antifungal drugs. Here we describe how antifungal susceptibility testing can be combined with target identification in distal ergosterol biosynthesis by means of gas chromatography-mass spectrometry...
May 2017: Nature Protocols
https://www.readbyqxmd.com/read/28384138/diverse-protocols-for-correlative-super-resolution-fluorescence-imaging-and-electron-microscopy-of-chemically-fixed-samples
#7
Benjamin G Kopek, Maria G Paez-Segala, Gleb Shtengel, Kem A Sochacki, Mei G Sun, Yalin Wang, C Shan Xu, Schuyler B van Engelenburg, Justin W Taraska, Loren L Looger, Harald F Hess
Our groups have recently developed related approaches for sample preparation for super-resolution imaging within endogenous cellular environments using correlative light and electron microscopy (CLEM). Four distinct techniques for preparing and acquiring super-resolution CLEM data sets for aldehyde-fixed specimens are provided, including Tokuyasu cryosectioning, whole-cell mount, cell unroofing and platinum replication, and resin embedding and sectioning. The choice of the best protocol for a given application depends on a number of criteria that are discussed in detail...
May 2017: Nature Protocols
https://www.readbyqxmd.com/read/28384137/preparation-of-molecularly-imprinted-polymers-specific-to-glycoproteins-glycans-and-monosaccharides-via-boronate-affinity-controllable-oriented-surface-imprinting
#8
Rongrong Xing, Shuangshou Wang, Zijun Bie, Hui He, Zhen Liu
Molecularly imprinted polymers (MIPs) are materials that are designed to be receptors for a template molecule (e.g., a protein). They are made by polymerizing the polymerizable reagents in the presence of the template; when the template is removed, the material can be used for many applications that would traditionally use antibodies. Thus, MIPs are biomimetic of antibodies and in this capacity have found wide applications, such as sensing, separation and diagnosis. However, many imprinting approaches are uncontrollable, and facile imprinting approaches widely applicable to a large variety of templates remain limited...
May 2017: Nature Protocols
https://www.readbyqxmd.com/read/28358394/long-read-chia-pet-for-base-pair-resolution-mapping-of-haplotype-specific-chromatin-interactions
#9
Xingwang Li, Oscar Junhong Luo, Ping Wang, Meizhen Zheng, Danjuan Wang, Emaly Piecuch, Jacqueline Jufen Zhu, Simon Zhongyuan Tian, Zhonghui Tang, Guoliang Li, Yijun Ruan
Chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) is a robust method for capturing genome-wide chromatin interactions. Unlike other 3C-based methods, it includes a chromatin immunoprecipitation (ChIP) step that enriches for interactions mediated by specific target proteins. This unique feature allows ChIA-PET to provide the functional specificity and higher resolution needed to detect chromatin interactions, which chromosome conformation capture (3C)/Hi-C approaches have not achieved. The original ChIA-PET protocol generates short paired-end tags (2 × 20 base pairs (bp)) to detect two genomic loci that are far apart on linear chromosomes but are in spatial proximity in the folded genome...
May 2017: Nature Protocols
https://www.readbyqxmd.com/read/28358393/on-chip-human-microvasculature-assay-for-visualization-and-quantification-of-tumor-cell-extravasation-dynamics
#10
Michelle B Chen, Jordan A Whisler, Julia Fröse, Cathy Yu, Yoojin Shin, Roger D Kamm
Distant metastasis, which results in >90% of cancer-related deaths, is enabled by hematogenous dissemination of tumor cells via the circulation. This requires the completion of a sequence of complex steps including transit, initial arrest, extravasation, survival and proliferation. Increased understanding of the cellular and molecular players enabling each of these steps is key to uncovering new opportunities for therapeutic intervention during early metastatic dissemination. As a protocol extension, this article describes an adaptation to our existing protocol describing a microfluidic platform that offers additional applications...
May 2017: Nature Protocols
https://www.readbyqxmd.com/read/28358392/analyzing-trapped-protein-complexes-by-virotrap-and-sfinx
#11
Kevin Titeca, Emmy Van Quickelberghe, Noortje Samyn, Delphine De Sutter, Annick Verhee, Kris Gevaert, Jan Tavernier, Sven Eyckerman
The analysis of protein interaction networks is one of the key challenges in the study of biology. It connects genotypes to phenotypes, and disruption of such networks is associated with many pathologies. Virtually all the approaches to the study of protein complexes require cell lysis, a dramatic step that obliterates cellular integrity and profoundly affects protein interactions. This protocol starts with Virotrap, a novel approach that avoids the need for cell homogenization by fusing the protein of interest to the HIV-1 Gag protein, trapping protein complexes in virus-like particles...
May 2017: Nature Protocols
https://www.readbyqxmd.com/read/28333915/directed-differentiation-of-human-induced-pluripotent-stem-cells-into-functional-cholangiocyte-like-cells
#12
Fotios Sampaziotis, Miguel Cardoso de Brito, Imbisaat Geti, Alessandro Bertero, Nicholas Rf Hannan, Ludovic Vallier
The difficulty in isolating and propagating functional primary cholangiocytes is a major limitation in the study of biliary disorders and the testing of novel therapeutic agents. To overcome this problem, we have developed a platform for the differentiation of human pluripotent stem cells (hPSCs) into functional cholangiocyte-like cells (CLCs). We have previously reported that our 26-d protocol closely recapitulates key stages of biliary development, starting with the differentiation of hPSCs into endoderm and subsequently into foregut progenitor (FP) cells, followed by the generation of hepatoblasts (HBs), cholangiocyte progenitors (CPs) expressing early biliary markers and mature CLCs displaying cholangiocyte functionality...
April 2017: Nature Protocols
https://www.readbyqxmd.com/read/28333914/genome-scale-crispr-cas9-knockout-and-transcriptional-activation-screening
#13
Julia Joung, Silvana Konermann, Jonathan S Gootenberg, Omar O Abudayyeh, Randall J Platt, Mark D Brigham, Neville E Sanjana, Feng Zhang
Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. Recently, the RNA-guided endonuclease Cas9 from the microbial CRISPR (clustered regularly interspaced short palindromic repeats) immune system has been adapted for genome-scale screening by combining Cas9 with pooled guide RNA libraries. Here we describe a protocol for genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 system...
April 2017: Nature Protocols
https://www.readbyqxmd.com/read/28301462/dissection-of-mechanical-force-in-living-cells-by-super-resolved-traction-force-microscopy
#14
Huw Colin-York, Christian Eggeling, Marco Fritzsche
Cells continuously exert or respond to mechanical force. Measurement of these nanoscale forces is a major challenge in cell biology; yet such measurement is essential to the understanding of cell regulation and function. Current methods for examining mechanical force generation either necessitate dedicated equipment or limit themselves to coarse-grained force measurements on the micron scale. In this protocol, we describe stimulated emission depletion traction force microscopy-STED-TFM (STFM), which allows higher sampling of the forces generated by the cell than conventional TFM, leading to a twofold increase in spatial resolution (of up to 500 nm)...
April 2017: Nature Protocols
https://www.readbyqxmd.com/read/28301461/metabolomics-and-lipidomics-using-traveling-wave-ion-mobility-mass-spectrometry
#15
Giuseppe Paglia, Giuseppe Astarita
Metabolomics and lipidomics aim to profile the wide range of metabolites and lipids that are present in biological samples. Recently, ion mobility spectrometry (IMS) has been used to support metabolomics and lipidomics applications to facilitate the separation and the identification of complex mixtures of analytes. IMS is a gas-phase electrophoretic technique that enables the separation of ions in the gas phase according to their charge, shape and size. Occurring within milliseconds, IMS separation is compatible with modern mass spectrometry (MS) operating with microsecond scan speeds...
April 2017: Nature Protocols
https://www.readbyqxmd.com/read/28277548/optimized-labeling-of-membrane-proteins-for-applications-to-super-resolution-imaging-in-confined-cellular-environments-using-monomeric-streptavidin
#16
Ingrid Chamma, Olivier Rossier, Grégory Giannone, Olivier Thoumine, Matthieu Sainlos
Recent progress in super-resolution imaging (SRI) has created a strong need to improve protein labeling with probes of small size that minimize the target-to-label distance, increase labeling density, and efficiently penetrate thick biological tissues. This protocol describes a method for labeling genetically modified proteins incorporating a small biotin acceptor peptide with a 3-nm fluorescent probe, monomeric streptavidin. We show how to express, purify, and conjugate the probe to organic dyes with different fluorescent properties, and how to label selectively biotinylated membrane proteins for SRI techniques (point accumulation in nanoscale topography (PAINT), stimulated emission depletion (STED), stochastic optical reconstruction microscopy (STORM))...
April 2017: Nature Protocols
https://www.readbyqxmd.com/read/28277547/backbone-assignment-of-perdeuterated-proteins-by-solid-state-nmr-using-proton-detection-and-ultrafast-magic-angle-spinning
#17
Pascal Fricke, Veniamin Chevelkov, Maximilian Zinke, Karin Giller, Stefan Becker, Adam Lange
Solid-state NMR (ssNMR) is a technique that allows the study of protein structure and dynamics at atomic detail. In contrast to X-ray crystallography and cryo-electron microscopy, proteins can be studied under physiological conditions-for example, in a lipid bilayer and at room temperature (0-35 °C). However, ssNMR requires considerable amounts (milligram quantities) of isotopically labeled samples. In recent years, (1)H-detection of perdeuterated protein samples has been proposed as a method of alleviating the sensitivity issue...
April 2017: Nature Protocols
https://www.readbyqxmd.com/read/28277546/decerebrate-mouse-model-for-studies-of-the-spinal-cord-circuits
#18
Claire F Meehan, Kyle A Mayr, Marin Manuel, Stan T Nakanishi, Patrick J Whelan
The adult decerebrate mouse model (a mouse with the cerebrum removed) enables the study of sensory-motor integration and motor output from the spinal cord for several hours without compromising these functions with anesthesia. For example, the decerebrate mouse is ideal for examining locomotor behavior using intracellular recording approaches, which would not be possible using current anesthetized preparations. This protocol describes the steps required to achieve a low-blood-loss decerebration in the mouse and approaches for recording signals from spinal cord neurons with a focus on motoneurons...
April 2017: Nature Protocols
https://www.readbyqxmd.com/read/28253237/translation-complex-profile-sequencing-to-study-the-in-vivo-dynamics-of-mrna-ribosome-interactions-during-translation-initiation-elongation-and-termination
#19
Nikolay E Shirokikh, Stuart K Archer, Traude H Beilharz, David Powell, Thomas Preiss
Messenger RNA (mRNA) translation is a tightly controlled process that is integral to gene expression. It features intricate and dynamic interactions of the small and large subunits of the ribosome with mRNAs, aided by multiple auxiliary factors during distinct initiation, elongation and termination phases. The recently developed ribosome profiling method can generate transcriptome-wide surveys of translation and its regulation. Ribosome profiling records the footprints of fully assembled ribosomes along mRNAs and thus primarily interrogates the elongation phase of translation...
April 2017: Nature Protocols
https://www.readbyqxmd.com/read/28253236/coordinated-generation-of-multiple-ocular-like-cell-lineages-and-fabrication-of-functional-corneal-epithelial-cell-sheets-from-human-ips-cells
#20
Ryuhei Hayashi, Yuki Ishikawa, Ryousuke Katori, Yuzuru Sasamoto, Yuki Taniwaki, Hiroshi Takayanagi, Motokazu Tsujikawa, Kiyotoshi Sekiguchi, Andrew J Quantock, Kohji Nishida
We describe a protocol for the generation of a functional and transplantable corneal epithelium derived from human induced pluripotent stem (iPS) cells. When this protocol is followed, a proportion of iPS cells spontaneously form circular colonies, each of which is composed of four concentric zones. Cells in these zones have different morphologies and immunostaining characteristics, resembling neuroectoderm, neural crest, ocular-surface ectoderm, or surface ectoderm. We have named this 2D colony a 'SEAM' (self-formed ectodermal autonomous multizone), and previously demonstrated that cells within the SEAM have the potential to give rise to anlages of different ocular lineages, including retinal cells, lens cells, and ocular-surface ectoderm...
April 2017: Nature Protocols
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