Singamaneni研究室と共同研究者は、細胞からのタンパク質分泌を検出する新しいアッセイを開発しました。 Singamaneni’s lab, collaborators develop new assay that detects protein secretion from cells
2022-08-05 ワシントン大学セントルイス
FluoroDOTアッセイを開発し、8月5日に『Cell Reports Methods』誌で発表した。この高感度アッセイは、1つの細胞から分泌されるタンパク質を約30分で確認・測定することができます。
FluoroDOTアッセイが汎用性が高く、低コストであらゆる実験環境に適応でき、広く使われている既存のアッセイよりもこれらのタンパク質をより包括的に見ることができる可能性があることを発見しました。
FluoroDOTアッセイが既存のアッセイと異なるのは、シンガメニの研究室で開発されたプラズモン増強ナノラベルであるプラズモン蛍光体を用いている点で、従来の蛍光ラベルに比べて1万6000倍の明るさと30倍近いS/N比を持つ。
<関連情報>
- https://source.wustl.edu/2022/08/simple-yet-powerful-seeing-cell-secretion-like-never-before/
- https://www.cell.com/cell-reports-methods/fulltext/S2667-2375(22)00144-8
プラズモン励起FluoroDOTアッセイを用いた単細胞レベルでのタンパク質分泌の高分解能イメージング High-resolution imaging of protein secretion at the single-cell level using plasmon-enhanced FluoroDOT assay
Anushree Seth,Ekansh Mitta,Jingyi Lua,Samhitha Koll,Monty B. Maze,Hemant Josh,Rohit Gupt,Priya Rath,Zheyu Wan,Jeremiah J. Morrisse,Joel D. Erns,Cynthia Portal-Celha,Sharon Celeste Morle.Jennifer A. Philips,Srikanth Singamaneni
Cell Reports Methods Published:August 05, 2022
DOI:https://doi.org/10.1016/j.crmeth.2022.100267
Highlights
•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
Motivation
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.
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.
