Journal: STAR Protocols

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Publisher

Cell Press

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ISSN

2666-1667

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2020 - 202514
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Publications 1 - 10 of 14
  • Defining the interactomes of proteins involved in cytoskeletal dynamics using high-throughput proximity-dependent biotinylation in cellulo
    Item type: Journal Article
    Nahlé, Sarah; Quirion, Laura; Boulais, Jonathan; et al. (2022)
    STAR Protocols
    Proximity-dependent biotinylation (BioID) screens are excellent tools to capture in cellulo interactomes for a large variety of baits, including transient and weak affinity interactions, as well as localization-specific proximity components, which are much harder to detect with conventional approaches. Here, we describe the major starting steps and a detailed protocol on how to perform BioID in mammalian cells. We also describe the mass spectrometry procedure and the bioinformatics pipeline for the data analysis. For complete details on the use and execution of this profile, please refer to Bagci et al. (2020).
  • scQUEST: Quantifying tumor ecosystem heterogeneity from mass or flow cytometry data
    Item type: Journal Article
    Martinelli, Adriano Luca; Wagner, Johanna; Bodenmiller, Bernd; et al. (2022)
    STAR Protocols
    With mass and flow cytometry, millions of single-cell profiles with dozens of parameters can be measured to comprehensively characterize complex tumor ecosystems. Here, we present scQUEST, an open-source Python library for cell type identification and quantification of tumor ecosystem heterogeneity in patient cohorts. We provide a step-by-step protocol on the application of scQUEST on our previously generated human breast cancer single-cell atlas using mass cytometry and discuss how it can be adapted and extended for other datasets and analyses. For complete details on the use and execution of this protocol, please refer to Wagner et al. (2019).
  • Mass spectrometry analysis of circulating breast cancer cells from a Xenograft mouse model
    Item type: Journal Article
    Donato, Cinzia; Buczak, Katarzyna; Schmidt, Alexander; et al. (2021)
    STAR Protocols
    Circulating tumor cells (CTCs) are precursors of metastasis in various cancer types. Many aspects regarding CTC biology remain poorly understood. Here, we describe mass spectrometric analysis of CTCs from a breast cancer xenograft mouse model, including procedures comprising CTC enrichment, separation of different CTC subpopulations, and their quantitative proteomic assessment. This protocol aims to facilitate the identification of protein content dynamics in human CTCs that are physiologically shed from tumor-bearing xenografts, providing a framework for investigating metastasis biology. For complete details on the use and execution of this protocol, please refer to Donato et al. (2020).
  • Fast In Vitro Procedure to Identify Extraembryonic Differentiation Defect of Mouse Embryonic Stem Cells
    Item type: Journal Article
    Ngondo, Richard P.; Cohen-Tannoudji, Michel; Ciaudo, Constance (2020)
    STAR Protocols
    Mouse embryonic stem cells (mESCs) are a powerful model to study early mouse development. These blastocyst-derived cells can maintain pluripotency and differentiate into the three embryonic germ layers and an extraembryonic layer, the extraembryonic endoderm (ExEn), which shares similar molecular markers to the definitive endoderm. Here, we present a fast procedure to identify a differentiation defect of mESCs toward ExEn in vitro through the molecular and cellular characterization of embryoid bodies, followed by direct differentiation of mESCs into ExEn.
  • Time-lapse and cleared imaging of mouse embryonic lung explants to study three-dimensional cell morphology and topology dynamics
    Item type: Journal Article
    Gómez, Harold Fernando; Doumpas, Nikolaos; Iber, Dagmar (2023)
    STAR Protocols
    Here, we present a protocol for collecting high-spatiotemporal-resolution datasets of undisturbed mouse embryonic epithelial rudiments using light-sheet fluorescence microscopy. We describe steps for rudiment dissection, clearing, and embedding for cleared and live imaging. We then detail procedures for light-sheet imaging followed by image processing and morphometric analysis. We provide protocol variations for imaging both growing and optically cleared lung explants to encourage the quantitative exploration of three-dimensional cell shapes, cell organization, and complex cell-cell dynamics. For complete details on the use and execution of this protocol, please refer to Gómez et al. (2021).1
  • Unraveling virus-induced cellular signaling cascades by label-free quantitative phosphoproteomics
    Item type: Journal Article
    Hunziker, Annika; Stertz, Silke (2022)
    STAR Protocols
    Due to the low stoichiometry and highly transient nature of protein phosphorylation it is challenging to capture the dynamics and complexity of phosphorylation events on a systems level. Here, we present an optimized protocol to measure virus-induced phosphorylation events with high sensitivity using label free quantification-based phosphoproteomics. Specifically, we describe filter assisted protein digestion (FASP), enrichment of phosphopeptides, mass spectrometry, and subsequent bioinformatic analysis. For complete details on the use and execution of this protocol, please refer to Hunziker et al. (2022).
  • Nuclear RNA purification by flow cytometry to study nuclear processes in plants
    Item type: Journal Article
    Moro, Belén; Kisielow, Malgorzata; Barragan Borrero, Veronica; et al. (2021)
    STAR Protocols
    The nature of plant tissues has continuously hampered understanding of the spatio-temporal and subcellular distribution of RNA-guided processes. Here, we describe a universal protocol based on Arabidopsis to investigate subcellular RNA distribution from virtually any plant species using flow cytometry sorting. This protocol includes all necessary control steps to assess the quality of the nuclear RNA purification. Moreover, it can be easily applied to different plant developmental stages, tissues, cell cycle phases, experimental growth conditions, and specific cell type(s). For complete information on the use and execution of this protocol, please refer to Bologna et al. (2018) and de Leone et al. (2020).
  • Quantification of adipocyte numbers in transgenic mice via the Cre-LoxP recombination sites
    Item type: Journal Article
    Moser, Caroline; Straub, Leon G.; Rachamin, Yael; et al. (2021)
    STAR Protocols
    This protocol describes a method to assess adipocyte numbers within a specific depot based on their inducible genomic label. By extracting DNA from a complete adipose tissue depot stemming from two transgenic mouse lines (Adipoq-CreERT2 x ROSA26-tdRFP and Ucp1-CreERT2 x ROSA26-tdRFP), the number of adipocytes can be determined based on the quantification of the recombined LoxPRed sites. This highly sensitive system allows for the quantification of white, brown, and brite/beige adipocytes in a spatially unbiased and size-independent manner. For complete details on the use and execution of this protocol, please refer to Moser et al. (2021).
  • Purification of Affinity Tag-free Recombinant Tubulin from Insect Cells
    Item type: Journal Article
    Ti, Shih-Chieh; Wieczorek, Michal; Kapoor, Tarun M. (2020)
    STAR Protocols
    α/β-tubulin heterodimers, which can harbor diverse isotypes and post-translational modifications, polymerize into microtubules that are fundamental to many cellular processes. Due to long-standing challenges in generating recombinant tubulin, however, it has been difficult to examine the properties of specific tubulin isotypes. Here, we provide a protocol for purifying milligrams of affinity tag-free, isotypically pure recombinant tubulin. Our method can be applicable to any tubulin of interest, opening the door to dissecting how tubulin diversity regulates microtubule function. For complete details on the use and execution of this protocol, please see Ti et al. (2018).
  • An optimized protocol for assessing changes in mouse whole-brain activity using opto-fMRI
    Item type: Journal Article
    Grimm, Christina; Wenderoth, Nicole; Zerbi, Valerio (2022)
    STAR Protocols
    Functional magnetic resonance imaging (fMRI) in mouse brain, paired with spatially and temporally defined manipulations, offers a powerful tool to causally explain the effect of specific neuronal activity on brain network dynamics. Here, we present an optimized protocol to measure cell-type-specific contributions to changes in whole-brain dynamics in mice using optogenetics (opto)-fMRI. This protocol details the injection of ChR2-expressing AAV, the implantation of optical fiber, the steps to perform opto-BOLD (blood-oxygenation-level-dependent) fMRI recording, and data analysis. For complete details on the use and execution of this protocol, please refer to Grimm et al. (2021).
Publications 1 - 10 of 14