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Quantifying Heterogeneity of Individual Organelles in Mixed Populations via Mass Cytometry

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    Quantifying Heterogeneity of Individual Organelles in Mixed Populations via Mass Cytometry
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    Analytical Chemistry

    Cite this: Anal. Chem. 2018, 90, 22, 13315–13321
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    https://doi.org/10.1021/acs.analchem.8b02790
    Published October 17, 2018
    Copyright © 2018 American Chemical Society
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    Abstract

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    Macroautophagy is a complex degradative intracellular process by which long-lived proteins and damaged organelles are cleared. Common methods for the analysis of autophagy are bulk measurements which mask organelle heterogeneity and complicate the analysis of interorganelle association and trafficking. Thus, methods for individual organelle quantification are needed to address these deficiencies. Current techniques for quantifying individual autophagy organelles are either low through-put or are dimensionally limited. We make use of the multiparametric capability of mass cytometry to investigate phenotypic heterogeneity in autophagy-related organelle types that have been isolated from murine brain, liver, and skeletal muscle. Detection and phenotypic classification of individual organelles were accomplished through the use of a lanthanide-chelating membrane stain and organelle-specific antibodies. Posthoc sample matrix background correction and nonspecific antibody binding corrections provide measures of interorganelle associations and heterogeneity. This is the first demonstration of multiparametric individual organelle analysis via mass cytometry. The method described here illustrates the potential for further investigation of the inherently complex interorganelle associations, trafficking, and heterogeneity present in most eukaryotic biological systems.

    Copyright © 2018 American Chemical Society

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    Supporting Information

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.8b02790.

    • Table S-1, Metal-labeled antibody panel characterization; Figure S-1, intra-assay precision assessed via technical replicates; Figure S-2, organelle event gating strategy; Figure S-3, low signal threshold calculations; Table S-2, calculated low signal threshold values; Figure S-4, characterization of the abundance of phenotypically unique organelle populations; Figure S-5, comparison of the number of specific marker antibodies bound to each organelle type; Figure S-6, multi-dimensional t-SNE analysis of organelle from liver tissue; and Figure S-7, multi-dimensional t-SNE analysis of organelles from skeletal muscle tissue (PDF)

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    Cited By

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    This article is cited by 10 publications.

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    Analytical Chemistry

    Cite this: Anal. Chem. 2018, 90, 22, 13315–13321
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acs.analchem.8b02790
    Published October 17, 2018
    Copyright © 2018 American Chemical Society
    Request reuse permissions

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