High-resolution imaging of protein secretion at the single-cell level using plasmon-enhanced FluoroDOT assay

Secreted proteins mediate essential physiological processes. With conventional assays, it is challenging to map the spatial distribution of proteins secreted by single cells, to study cell-to-cell heterogeneity in secretion, or to detect proteins of low abundance or incipient secretion. Here, we int...

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Published in:Cell reports methods 2022-08, Vol.2 (8), p.100267-100267, Article 100267
Main Authors: Seth, Anushree, Mittal, Ekansh, Luan, Jingyi, Kolla, Samhitha, Mazer, Monty B., Joshi, Hemant, Gupta, Rohit, Rathi, Priya, Wang, Zheyu, Morrissey, Jeremiah J., Ernst, Joel D., Portal-Celhay, Cynthia, Morley, Sharon Celeste, Philips, Jennifer A., Singamaneni, Srikanth
Format: Article
Language:English
Subjects:
cytokine secretion
Cytokines - metabolism
fluorescence imaging
Humans
Interleukin-1 beta
Macrophages
plasmonic nanoparticles
single-cell secretion
T-Lymphocytes - metabolism
tuberculosis
Tuberculosis - metabolism
Tumor Necrosis Factor-alpha
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Summary:Secreted proteins mediate essential physiological processes. With conventional assays, it is challenging to map the spatial distribution of proteins secreted by single cells, to study cell-to-cell heterogeneity in secretion, or to detect proteins of low abundance or incipient secretion. Here, we introduce the “FluoroDOT assay,” which uses an ultrabright nanoparticle plasmonic-fluor that enables high-resolution imaging of protein secretion. We find that plasmonic-fluors are 16,000-fold brighter, with nearly 30-fold higher signal-to-noise compared with conventional fluorescence labels. We demonstrate high-resolution imaging of different secreted cytokines in the single-plexed and spectrally multiplexed FluoroDOT assay that revealed cellular heterogeneity in secretion of multiple proteins simultaneously. Using diverse biochemical stimuli, including Mycobacterium tuberculosis infection, and a variety of immune cells such as macrophages, dendritic cells (DCs), and DC-T cell co-culture, we demonstrate that the assay is versatile, facile, and widely adaptable for enhancing biological understanding of spatial and temporal dynamics of single-cell secretome. [Display omitted] •FluoroDOT is an ultrasensitive assay for studying protein secretion dynamics of single cells•FluoroDOT provide a digital, quantifiable signal for measuring cell-to-cell heterogeneity•We demonstrate applicability on wide-ranging cell-types and stimuli•A multicolor plasmonic-fluor palette allows multiplexed detection of secreted proteins Understanding the spatial and temporal dynamics of protein secretion at a single-cell level is essential to understanding numerous biological systems relevant to health and disease. However, currently available methods for studying protein secretion at the single-cell level lack spatial resolution. High-resolution imaging of single-cell secretion typically requires expensive and specialized microscopy facilities that are not routinely available. We developed and validated a simple yet powerful method called the “FluoroDOT” assay to image the spatial distribution of secreted proteins around single cells, along with the capability to quantify cell-to-cell heterogeneity in secretion. The ultrabright nanolabels enable the quantification of the signals in a digital manner. Understanding the spatial and temporal dynamics of protein secretion at a single-cell level is essential, but up until now remained challenging. To address this challenge, Seth et al. developed and
ISSN:2667-2375
2667-2375
DOI:10.1016/j.crmeth.2022.100267