Multiplexed single-cell analysis of organoid signaling networks.

Autor: Sufi J; Cell Communication Lab, Department of Oncology, University College London Cancer Institute, London, UK., Qin X; Cell Communication Lab, Department of Oncology, University College London Cancer Institute, London, UK., Rodriguez FC; Cell Communication Lab, Department of Oncology, University College London Cancer Institute, London, UK., Bu YJ; Department of Chemistry, University of Toronto, Toronto, Ontario, Canada., Vlckova P; Cell Communication Lab, Department of Oncology, University College London Cancer Institute, London, UK., Zapatero MR; Cell Communication Lab, Department of Oncology, University College London Cancer Institute, London, UK., Nitz M; Department of Chemistry, University of Toronto, Toronto, Ontario, Canada., Tape CJ; Cell Communication Lab, Department of Oncology, University College London Cancer Institute, London, UK. c.tape@ucl.ac.uk.
Jazyk: angličtina
Zdroj: Nature protocols [Nat Protoc] 2021 Oct; Vol. 16 (10), pp. 4897-4918. Date of Electronic Publication: 2021 Sep 08.
DOI: 10.1038/s41596-021-00603-4
Abstrakt: Organoids are biomimetic tissue models comprising multiple cell types and cell states. Post-translational modification (PTM) signaling networks control cellular phenotypes and are frequently dysregulated in diseases such as cancer. Although signaling networks vary across cell types, there are limited techniques to study cell type-specific PTMs in heterocellular organoids. Here, we present a multiplexed mass cytometry (MC) protocol for single-cell analysis of PTM signaling and cell states in organoids and organoids co-cultured with fibroblasts and leukocytes. We describe how thiol-reactive organoid barcoding in situ (TOBis) enables 35-plex and 126-plex single-cell comparison of organoid cultures and provide a cytometry by time of flight (CyTOF) signaling analysis pipeline (CyGNAL) for computing cell type-specific PTM signaling networks. The TOBis MC protocol takes ~3 d from organoid fixation to data acquisition and can generate single-cell data for >40 antibodies from millions of cells across 126 organoid cultures in a single MC run.
(© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)
Databáze: MEDLINE
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