| 1. |
- Bojmar, Linda, 1983-, et al.
(författare)
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Extracellular vesicle and particle isolation from human and murine cell lines, tissues, and bodily fluids
- 2021
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Ingår i: STAR protocols. - : Cell Press. - 2666-1667. ; 2:1
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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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| 2. |
- Boutet-Robinet, Elisa, et al.
(författare)
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Detection of DNA damage by alkaline comet assay in mouse colonic mucosa
- 2021
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Ingår i: STAR Protocols. - : Cell Press. - 2666-1667. ; 2:4
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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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| 6. |
- Generó, Magalí Martí, 1983-, et al.
(författare)
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A protocol for characterization of extremely preterm infant gut microbiota in double-blind clinical trials
- 2021
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Ingår i: STAR Protocols. - Cambridge, MA, United States : Cell press. - 2666-1667. ; 2:3
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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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| 7. |
- Kawale, Ashish A., et al.
(författare)
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Characterization of backbone dynamics using solution NMR spectroscopy to discern the functional plasticity of structurally analogous proteins
- 2021
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Ingår i: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 2:4
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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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| 8. |
- Kuznetsova, Irina, et al.
(författare)
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OmicsVolcano : Software for intuitive visualization and interactive exploration of high-throughput biological data
- 2021
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Ingår i: STAR Protocols. - : Elsevier. - 2666-1667. ; 2:1
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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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| 9. |
- Lopez Chiloeches, Maria, et al.
(författare)
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Characterization of macrophage infiltration and polarization by double fluorescence immunostaining in mouse colonic mucosa
- 2021
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Ingår i: STAR Protocols. - : Cell Press. - 2666-1667. ; 2:4
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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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| 10. |
- Park, Se Hyung, et al.
(författare)
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A luminescence-based protocol for assessing fructose metabolism via quantification of ketohexokinase enzymatic activity in mouse or human hepatocytes
- 2021
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Ingår i: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 2:3
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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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| 11. |
- Richardson, Simon E., et al.
(författare)
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In vitro differentiation of human pluripotent stem cells into the B lineage using OP9-MS5 co-culture
- 2021
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Ingår i: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 2:2
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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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| 12. |
- Santoro, Federica, et al.
(författare)
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Isolation of human ESC-derived cardiac derivatives and embryonic heart cells for population and single-cell RNA-seq analysis
- 2021
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Ingår i: STAR PROTOCOLS. - : Elsevier. - 2666-1667. ; 2:1
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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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