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2.
  • Al-Jubair, Tamim, et al. (författare)
  • Characterization of human aquaporin protein-protein interactions using microscale thermophoresis (MST)
  • 2022
  • Ingår i: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 3:2
  • Tidskriftsartikel (refereegranskat)abstract
    • Aquaporin water channels (AQPs) are membrane proteins that maintain cellular water homeostasis. The interactions between human AQPs and other proteins play crucial roles in AQP regulation by both gating and trafficking. Here, we describe a protocol for characterizing the interaction between a human AQP and a soluble interaction partner using microscale thermophoresis (MST). MST has the advantage of low sample consumption and high detergent compatibility enabling AQP protein-protein interaction investigation with a high level of control of components and environment. For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020) and Roche et al. (2017).
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3.
  • Al-Jubair, Tamim, et al. (författare)
  • High-yield overproduction and purification of human aquaporins from Pichia pastoris
  • 2022
  • Ingår i: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 3:2
  • Tidskriftsartikel (refereegranskat)abstract
    • Aquaporins (AQPs) are membrane-bound water channels that play crucial roles in maintaining the water homeostasis of the human body. Here, we present a protocol for high-yield recombinant expression of human AQPs in the methylotropic yeast Pichia pastoris and subsequent AQP purification. The protocol typically yields 1–5 mg AQP per g of yeast cell at >95% purity and is compatible with any membrane protein cloned into Pichia pastoris, although expression levels may vary. For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020) and Frick et al. (2014).
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  • Arellano-Caicedo, Carlos, et al. (författare)
  • Quantification of growth and nutrient consumption of bacterial and fungal cultures in microfluidic microhabitat models
  • 2024
  • Ingår i: STAR Protocols. - 2666-1667. ; 5:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Understanding microbes in nature requires consideration of their microenvironment. Here, we present a protocol for quantifying biomass and nutrient degradation of bacterial and fungal cultures (Pseudomonas putida and Coprinopsis cinerea, respectively) in microfluidics. We describe steps for mask design and fabrication, master printing, polydimethylsiloxane chip fabrication, and chip inoculation and imaging using fluorescence microscopy. We include procedures for image analysis, plotting, and statistics. For complete details on the use and execution of this protocol, please refer to Arellano-Caicedo et al. (2023).1
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  • Bojmar, Linda, 1983-, et al. (författare)
  • Extracellular vesicle and particle isolation from human and murine cell lines, tissues, and bodily fluids
  • 2021
  • Ingår i: STAR protocols. - : Cell Press. - 2666-1667. ; 2:1
  • Tidskriftsartikel (refereegranskat)abstract
    • We developed a modified protocol, based on differential ultracentrifugation (dUC), to isolate extracellular vesicles and particles (specifically exomeres) (EVPs) from various human and murine sources, including cell lines, surgically resected tumors and adjacent tissues, and bodily fluids, such as blood, lymphatic fluid, and bile. The diversity of these samples requires robust and highly reproducible protocols and refined isolation technology, such as asymmetric-flow field-flow fractionation (AF4). Our isolation protocol allows for preparation of EVPs for various downstream applications, including proteomic profiling. For complete details on the use and execution of this protocol, please refer to Hoshino et al. (2020).
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8.
  • Bojmar, Linda, et al. (författare)
  • Protocol for cross-platform characterization of human and murine extracellular vesicles and particles
  • 2024
  • Ingår i: STAR PROTOCOLS. - : ELSEVIER. - 2666-1667. ; 5:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Characterization of isolated extracellular vesicles and particles (EVPs) is crucial for determining functions and biomarker potential. Here, we present a protocol to analyze size, number, morphology, and EVP protein cargo and to validate EVP proteins in both humans and mice. We describe steps for nanoparticle tracking analysis, transmission electron microscopy, single-EVP immunodetection, EVP proteomic mass spectrometry and bioinformatic analysis, and EVP protein validation by ExoELISA and western blot analysis. This allows for EVP cross -validation across different platforms.For complete details on the use and execution of this protocol, please refer to Hoshino et al.1
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  • Boutet-Robinet, Elisa, et al. (författare)
  • Detection of DNA damage by alkaline comet assay in mouse colonic mucosa
  • 2021
  • Ingår i: STAR Protocols. - : Cell Press. - 2666-1667. ; 2:4
  • Tidskriftsartikel (refereegranskat)abstract
    • We recently characterized the association between DNA damage and immunoresponse in vivo in colonic mucosa of mice infected with a Salmonella Typhimurium strain expressing a genotoxin, known as typhoid toxin. In this protocol, we describe the specific steps for assessing DNA damage by the alkaline comet assay of colonic mucosal samples. The description of the comet assay protocol follows the international guidelines (Minimum Information for Reporting on the Comet Assay [Moller et al., 2020]). For complete details on the use and execution of this protocol, please refer to Martin et al. (2021).
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11.
  • Bronnec, Vicky, et al. (författare)
  • Detailed protocol for germ-free Drosophila melanogaster colonization with Propionibacterium spp. biofilms
  • 2022
  • Ingår i: STAR Protocols. - : Cell Press. - 2666-1667. ; 3:2
  • Tidskriftsartikel (refereegranskat)abstract
    • In this protocol, we describe a germ-free Drosophila melanogaster model to investigate anaerobic bacterial biofilms. We detail how to establish Propionibacterium spp. biofilms in the fruit fly's gut using an easy to carry out method. For complete details on the use and execution of this protocol, please refer to Bronnec and Alexeyev (2021) and Bronnec et al. (2022).
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  • Erttmann, Saskia F., et al. (författare)
  • Protocol for isolation of microbiota-derived membrane vesicles from mouse blood and colon
  • 2023
  • Ingår i: STAR Protocols. - : CellPress. - 2666-1667. ; 4:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Bacterial membrane vesicles have emerged as gadgets allowing remote communication between the microbiota and distal host organs. Here we describe a protocol for enriching vesicles from serum and colon that could widely be adapted for other tissues. We detail pre-clearing of serum or colon fluids using 0.2-μm syringe filters and their concentration by centrifugal filter devices. We also describe vesicle isolation with qEV size exclusion columns and finally the concentration of isolated vesicle fractions for downstream analyses. For complete details on the use and execution of this protocol, please refer to Erttmann et al. (2022).1
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  • Generó, Magalí Martí, 1983-, et al. (författare)
  • A protocol for characterization of extremely preterm infant gut microbiota in double-blind clinical trials
  • 2021
  • Ingår i: STAR Protocols. - Cambridge, MA, United States : Cell press. - 2666-1667. ; 2:3
  • Tidskriftsartikel (refereegranskat)abstract
    • 16S rRNA gene sequencing enables microbial community profiling, but recovering fecal DNA from extremely premature infants is challenging. Here, we describe an optimized protocol for fecal DNA isolation, library preparation for 16S rRNA gene sequencing, taxonomy assignation, and statistical analyses. The protocol is complemented with a quantitative PCR for probiotic L. reuteri identification. This protocol describes how to characterize preterm infant gut microbiota and relate it to probiotic supplementation and clinical outcomes. It is customizable for other clinical trials. For complete details on the use and execution of this protocol, please refer to Martí et al. (2021) and Spreckels et al. (2021).
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19.
  • Giacomoni, Jessica, et al. (författare)
  • Protocol for optical clearing and imaging of fluorescently labeled ex vivo rat brain slices
  • 2023
  • Ingår i: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 4:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Tissue clearing is commonly used for whole-brain imaging but seldom used for brain slices. Here, we present a simple protocol to slice, immunostain, and clear sections of adult rat brains for subsequent high-resolution confocal imaging. The protocol does not require toxic reagents or specialized equipment. We also provide instructions for culturing of rat brain slices free floating on permeable culture inserts, maintained in regular CO2 incubators, and handled only at media change.
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  • Ignatov, Dmitriy, et al. (författare)
  • Generation of Sequencing Libraries for Structural Analysis of Bacterial 5′ UTRs
  • 2020
  • Ingår i: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 1:2
  • Tidskriftsartikel (refereegranskat)abstract
    • The structure of 5′ untranslated regions (5′ UTRs) of bacterial mRNAs often determines the fate of the transcripts. Using a dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) approach, we developed a protocol to generate sequence libraries to determine the base-pairing status of adenines and cytosines in the 5′ UTRs of bacterial mRNAs. Our method increases the sequencing depth of the 5′ UTRs and allows detection of changes in their structures by sequencing libraries of moderate sizes. For complete details on the use and execution of this protocol, please refer to Ignatov et al. (2020).
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  • Johansson, Pia Annette, et al. (författare)
  • CRISPRi-mediated transcriptional silencing in iPSCs for the study of human brain development
  • 2022
  • Ingår i: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 3:2
  • Tidskriftsartikel (refereegranskat)abstract
    • This protocol describes the design and use of CRISPRi-mediated transcriptional silencing in human iPSCs, for loss-of-function studies in brain development research. The protocol avoids single cell selection, thereby eliminating side effects of clonal expansion and sites of viral integration. We also describe a neural progenitor differentiation protocol and discuss the challenges of target-specific lentiviral silencing, efficient silencing levels, and off-target effects. For complete details on the use and execution of this protocol, please refer to Johansson et al. (2022).
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24.
  • Kawale, Ashish A., et al. (författare)
  • Characterization of backbone dynamics using solution NMR spectroscopy to discern the functional plasticity of structurally analogous proteins
  • 2021
  • Ingår i: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 2:4
  • Tidskriftsartikel (refereegranskat)abstract
    • The comprehensive delineation of inherent dynamic motions embedded in proteins, which can be crucial for their functional repertoire, is often essential yet remains poorly understood in the majority of cases. In this protocol, we outline detailed descriptions of the necessary steps for employing solution NMR spectroscopy for the in-depth amino acid level understanding of backbone dynamics of proteins. We describe the application of the protocol on the structurally analogous Tudor domains with disparate functionalities as a model system. For complete details on the use and execution of this protocol, please refer to Kawale and Burmann (2021). © 2021 The Author(s)
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25.
  • Kitchen, Philip, et al. (författare)
  • Calcein Fluorescence Quenching to Measure Plasma Membrane Water Flux in Live Mammalian Cells
  • 2020
  • Ingår i: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 1:3
  • Tidskriftsartikel (refereegranskat)abstract
    • Aquaporins (AQPs) are membrane channel proteins that facilitate the movement of water down osmotic gradients across biological membranes. This protocol allows measurements of AQP-mediated water transport across the plasma membrane of live mammalian cells. Calcein is a fluorescent dye that is quenched in a concentration-dependent manner. Therefore, on short timescales, its concentration-dependent fluorescence can be used as a probe of cell volume, and therefore a probe of water transport into or out of cells. For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020) and Kitchen and Conner (2015). For the underlying methodology development, please refer to Fenton et al. (2010) and Solenov et al. (2004).
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27.
  • Kuznetsova, Irina, et al. (författare)
  • OmicsVolcano : Software for intuitive visualization and interactive exploration of high-throughput biological data
  • 2021
  • Ingår i: STAR Protocols. - : Elsevier. - 2666-1667. ; 2:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Advances in omics technologies have generated exponentially larger volumes of biological data; however, their analyses and interpretation are limited to computationally proficient scientists. We created OmicsVolcano, an interactive open-source software tool to enable visualization and exploration of high-throughput biological data, while highlighting features of interest using a volcano plot interface. In contrast to existing tools, our software and user-interface design allow it to be used without requiring any programming skills to generate high-quality and presentation-ready images.
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28.
  • Linde, Erika, et al. (författare)
  • A one-pot cascade protocol for diarylation of amines and water
  • 2022
  • Ingår i: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 3:4
  • Tidskriftsartikel (refereegranskat)abstract
    • N- and O-arylated compounds are prevalent in pharmaceuticals and materials, and efficient approaches for their synthesis are important. Herein, we present an efficient protocol for the diarylation of aliphatic amines and water with two structurally different aryl groups in one single step, yielding highly functionalized diaryl amines and ethers. We describe the synthesis of the required diaryliodonium salts and detail the procedure for the diarylation. The protocol is limited to use of unhindered amines and diaryliodonium salts with certain substituents.
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29.
  • Lopez Chiloeches, Maria, et al. (författare)
  • Characterization of macrophage infiltration and polarization by double fluorescence immunostaining in mouse colonic mucosa
  • 2021
  • Ingår i: STAR Protocols. - : Cell Press. - 2666-1667. ; 2:4
  • Tidskriftsartikel (refereegranskat)abstract
    • We recently characterized the association between DNA damage and immunoresponse in vivo in colonic mucosa of mice infected with a Salmonella Typhimurium strain expressing a genotoxin, known as typhoid toxin. In this protocol, we describe how to assess the extent and features of infiltrating macrophages by double immunofluorescence. Total macrophage population was determined using an F4/80 antibody, whereas the specific M2-like population was assessed using a CD206 antibody. For complete details on the use and execution of this protocol, please refer to Martin et al. (2021).
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  • Park, Se Hyung, et al. (författare)
  • A luminescence-based protocol for assessing fructose metabolism via quantification of ketohexokinase enzymatic activity in mouse or human hepatocytes
  • 2021
  • Ingår i: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 2:3
  • Tidskriftsartikel (refereegranskat)abstract
    • Ketohexokinase (KHK) catalyzes the first step of fructose metabolism. Inhibitors of KHK enzymatic activity are being evaluated in clinical trials for the treatment of non-alcoholic fatty liver disease (NAFLD) and diabetes. Here, we present a luminescence-based protocol to quantify KHK activity. The accuracy of this technique has been validated using knockdown and overexpression of KHK in vivo and in vitro. The specificity of the assay has been verified using 3-O-methyl-D-fructose, a non-metabolizable analog of fructose, heat inactivation of hexokinases, and depletion of potassium. For complete details on the use of this protocol, please refer to Damen et al. (2021).
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  • Rabenius, Adelina, et al. (författare)
  • Quantifying RNA synthesis at rate-limiting steps of transcription using nascent RNA-sequencing data
  • 2022
  • Ingår i: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 3:1, s. 101036-
  • Tidskriftsartikel (refereegranskat)abstract
    • Nascent RNA-sequencing tracks transcription at nucleotide resolution. The genomic distribution of engaged transcription complexes, in turn, uncovers functional genomic regions. Here, we provide analytical steps to (1) identify transcribed regulatory elements de novo genome-wide, (2) quantify engaged transcription complexes at enhancers, promoter-proximal regions, divergent transcripts, gene bodies, and termination windows, and (3) measure distribution of transcription machineries and regulatory proteins across functional genomic regions. This protocol tracks engaged transcription complexes across functional genomic regions demonstrated in human K562 erythroleukemia cells. For complete details on the use and execution of this protocol, please refer to Vihervaara et al. (2021).
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34.
  • Rezaei, Shiva, et al. (författare)
  • GBS-MeDIP : A protocol for parallel identification of genetic and epigenetic variation in the same reduced fraction of genomes across individuals
  • 2022
  • Ingår i: STAR protocols. - : Cell Press. - 2666-1667. ; 3:1
  • Tidskriftsartikel (refereegranskat)abstract
    • The GBS-MeDIP protocol combines two previously described techniques, Geno-type-by-Sequencing (GBS) and Methylated-DNA- Immunoprecipitation (MeDIP). Our method allows for parallel and cost-efficient interrogation of genetic and methylomic variants in the DNA of many reduced genomes, taking advantage of the barcoding of DNA samples performed in the GBS and the subsequent creation of DNA pools, then used as an input for the MeDIP. The GBS-MeDIP is particularly suitable to identify genetic and methylomic biomarkers when resources for whole genome interrogation are lacking.
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35.
  • Rezaei, Shiva, et al. (författare)
  • GBS-MeDIP: A protocol for parallel identification of genetic and epigenetic variation in the same reduced fraction of genomes across individuals
  • 2022
  • Ingår i: STAR Protocols. - : Cell Press. - 2666-1667. ; 3:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Summary:The GBS-MeDIP protocol combines two previously described techniques, Genotype-by-Sequencing (GBS) and Methylated-DNA-Immunoprecipitation (MeDIP). Our method allows for parallel and cost-efficient interrogation of genetic and methylomic variants in the DNA of many reduced genomes, taking advantage of the barcoding of DNA samples performed in the GBS and the subsequent creation of DNA pools, then used as an input for the MeDIP. The GBS-MeDIP is particularly suitable to identify genetic and methylomic biomarkers when resources for whole genome interrogation are lacking.
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36.
  • Richardson, Simon E., et al. (författare)
  • In vitro differentiation of human pluripotent stem cells into the B lineage using OP9-MS5 co-culture
  • 2021
  • Ingår i: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 2:2
  • Tidskriftsartikel (refereegranskat)abstract
    • In vitro differentiation of human pluripotent stem cells (hPSCs) offers a genetically tractable system to examine the physiology and pathology of human tissue development and differentiation. We have used this approach to model the earliest stages of human B lineage development and characterize potential target cells for the in utero initiation of childhood B acute lymphoblastic leukemia. Herein, we detail critical aspects of the protocol including reagent validation, controls, and examples of surface markers used for analysis and cell sorting. For complete details on the use and execution of this protocol, please refer to Boiers et al. (2018).
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  • Rzepka, Magdalena, 1992, et al. (författare)
  • Incorporation of reporter genes into mitochondrial DNA in budding yeast
  • 2022
  • Ingår i: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 3:2
  • Tidskriftsartikel (refereegranskat)abstract
    • Many aspects of mitochondrial gene expression are still unknown, which can be attributed to limitations in molecular tools. Here, we present a protocol to introduce reporter genes into the mitochondrial genome of budding yeast, Saccharomyces cerevisiae. Mitochondrially encoded reporter constructs can be used to interrogate various aspects of mitochondrial gene expression. The power of this technique is exemplified by a mitochondrially encoded nanoluciferase, which allows to monitor levels of mitochondrial translation under a variety of growth conditions.
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39.
  • Saavedra Guillem, Beatriz, et al. (författare)
  • Selective quantitative N-functionalization of unprotected α-amino acids using NHC-Ir(III) catalyst
  • 2023
  • Ingår i: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 4:2
  • Tidskriftsartikel (refereegranskat)abstract
    • Unnatural amino acids are valuable building blocks with numerous applications. Here, we present a quantitative technique for accessing mono-N-functionalized amino acids directly from unprotected substrates using alcohols as alkylating agents and an NHC-Ir(III) catalyst. We detail specific steps for catalyst preparation and application, as well as for catalyst recycling. The protocol excludes a few amino acids (l-cysteine, l-lysine, and l-arginine) and secondary alcohols.
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40.
  • Safi, Fatemeh, et al. (författare)
  • In vitro clonal multilineage differentiation of distinct murine hematopoietic progenitor populations
  • 2023
  • Ingår i: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 4:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Here we describe an in vitro co-culture system that can differentiate hematopoietic progenitor populations to all major hematopoietic lineages at clonal level. We present both a sensitive single-cell switch-culture system as well as a less laborious alternative barcoding protocol more convenient for larger cell numbers. Importantly, generation of all lineages from single long-term hematopoietic stem cells are described, following 21 days of culture. This protocol represents an efficient tool for validation experiments for single-cell genomics data. For complete details on the use and execution of this protocol, please refer to Safi et al. (2022).1
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41.
  • Salvatori, Roger, 1988, et al. (författare)
  • Mapping protein networks in yeast mitochondria using proximity-dependent biotin identification coupled to proteomics
  • 2020
  • Ingår i: STAR PROTOCOLS. - : Elsevier BV. - 2666-1667. ; 1:3
  • Tidskriftsartikel (refereegranskat)abstract
    • Proximity-dependent biotin identification (BioID) permits biotinylation of proteins interacting directly, indirectly, or just localized in proximity of a protein of interest (bait). Here, we describe how BioID coupled to proteomics and network biology can be used to map protein proximities in yeast mitochondria, aiding in visualization of complex protein-protein interaction landscapes. For complete information on the use and execution of this protocol, please refer to Singh et al., 2020.
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  • Santoro, Federica, et al. (författare)
  • Isolation of human ESC-derived cardiac derivatives and embryonic heart cells for population and single-cell RNA-seq analysis
  • 2021
  • Ingår i: STAR PROTOCOLS. - : Elsevier. - 2666-1667. ; 2:1
  • Tidskriftsartikel (refereegranskat)abstract
    • The combination of population and single-cell RNA sequencing analysis using hu-man embryonic stem cell (hESC) differentiation and developmental tissues is a powerful approach to elucidate an organ-specific cellular and molecular atlas in human embryogenesis. This protocol describes (1) cardiac-directed differentia-tion and isolation of hESC-derived cardiac derivatives with fluorescence -acti-vated cell sorting, (2) isolation of human embryonic heart-derived single cardiac cells, and (3) construction of cDNA libraries with Smart-seq2. These allow for the preparation of human developmental samples for comprehensive transcriptional analysis. For complete details on the use and execution of this protocol, please refer to Sahara et al. (2019).
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44.
  • Steffen, Jonas Hyld, et al. (författare)
  • Assessing water permeability of aquaporins in a proteoliposome-based stopped-flow setup
  • 2022
  • Ingår i: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 3:2
  • Tidskriftsartikel (refereegranskat)abstract
    • Aquaporins (AQPs) are water channels embedded in the cell membrane that are critical in maintaining water homeostasis. We describe a protocol for determining the water permeation capacity of AQPs reconstituted into proteoliposomes. Using a stopped-flow setup, AQP embedded in proteoliposomes are exposed to an osmogenic gradient that triggers water flux. The consequent effects on proteoliposome size can be tracked using the fluorescence of an internalized fluorophore. This enables controlled characterization of water flux by AQPs. For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020).
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45.
  • Wali, Gautam, et al. (författare)
  • Generation of human-induced pluripotent-stem-cell-derived cortical neurons for high-throughput imaging of neurite morphology and neuron maturation
  • 2023
  • Ingår i: STAR Protocols. - 2666-1667. ; 4:2
  • Tidskriftsartikel (refereegranskat)abstract
    • High-throughput imaging allows in vitro assessment of neuron morphology for screening populations under developmental, homeostatic, and/or disease conditions. Here, we present a protocol to differentiate cryopreserved human cortical neuronal progenitors into mature cortical neurons for high-throughput imaging analysis. We describe the use of a notch signaling inhibitor to generate homogeneous neuronal populations at densities amenable to individual neurite identification. We detail neurite morphology assessment via measuring multiple parameters including neurite length, branches, roots, segments and extremities, and neuron maturation.
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48.
  • Zou, Yatao, et al. (författare)
  • Protocol for efficient and self-healing near-infrared perovskite light-emitting diodes.
  • 2022
  • Ingår i: STAR protocols. - : Cell Press. - 2666-1667. ; 3:3
  • Tidskriftsartikel (refereegranskat)abstract
    • Preparation of highly efficient and stable perovskite light-emitting diodes (PeLEDs) with reproducible device performance is challenging. This protocol describes steps for fabrication of high-performance and self-healing PeLEDs. These include instructions for synthesis of charge-transporting zinc oxide (ZnO) nanocrystals, step-by-step device fabrication, and control over self-healing of the degraded devices. For complete details on the use and execution of this protocol, please refer to Teng et al. (2021).
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