Tuesday, March 21, 2023

Researchers develop new method to visualize single-cell protein secretion with surprising resolution

We’ve recently seen stunning images of distant galaxies revealed by the James Webb Telescope, previously only visible as faint spots. Researchers at Washington University in St. Louis have developed a new method to visualize proteins secreted by cells with astonishing resolution, making it the James Webb variant for visualizing single-cell protein secretion.

Srikant Singamaneni, a professor of mechanical engineering and materials science at the McKelvey School of Engineering, was led by Lillian and E. Researchers led by Lisle Hughes and Anushree Seth, a former postdoctoral scholar in Singamaneni’s lab, developed the Fluorodot assay, which they introduced. A paper in the journal Cell Reports Methods Aug. 5. The highly sensitive assay is able to visualize and measure the proteins secreted by a single cell in approximately 30 minutes.

In collaboration with researchers from the Washington University School of Medicine and other universities, they found that the FluoroDot assay is versatile, low-cost, and adaptable to any laboratory setting and is more widely used on these proteins than existing assays. ability to provide. , Biomedical researchers look to these secreted proteins for information on cell-to-cell communication, cell signaling, activation and inflammation, among other actions, but current methods are limited in sensitivity and the process can take up to 24 hours.

What makes the FluoroDOT assay different from existing assays is that it uses a plasmonic-fluor, a plasmon-enhanced nanolabel developed in Singamani’s lab that is 16,000 times brighter than conventional fluorescence labels and has a signal-to-low signal-to-fluor signal. -Noise ratio is about 30 times higher.

Plasmonic-floors are made of metal nanoparticles that serve as antennae to pull in light and enhance the fluorescence emission of molecular fluorophores, thus making it an ultrabright nanoparticle.”

Srikanth Singamaneni, Lillian and E. Lisle Hughes Professor of Mechanical Engineering and Materials Science, McKelvey School of Engineering

This ultrabright emission of plasmonic-fluor allows the user to see very small amounts of secreted proteins, which they are unable to do in current assays, and high-resolution signals to digitally capture the number of particles, or dot patterns, per cluster. measure using . Or using Spot, a custom-built algorithm. In addition, it does not require special equipment. Singamaneni and colleagues first published their work with plasmonic-floor in 2020 in Nature Biomedical Engineering.

The patent-pending plasmonic floor technology has been licensed to Oragent Bioscience LLC by the Office of Technology Management at Washington University in St. Louis.

“Using a simple fluorescence microscope, we are able to simultaneously image a cell with the spatial distribution of secreted proteins around it and now have cellular applications for organic bioscience,” said Seth, who works in Singamaneni’s lab. is a leading scientist. “We saw interesting secretion patterns for different cell types. This assay also enables concurrent visualization of the two types of proteins from different cells. When multiple cells are subjected to the same stimulus, we can differentiate cells that are secreting two proteins into a single one. time from those that are secreting only one protein or not secreting it at all.”

To validate the technology, the team used proteins secreted from both human and mouse cells, including immune cells infected with Mycobacterium tuberculosis.

One of the colleagues and co-authors, Jennifer A. Phillips, MD, PhD, Theodore and Bertha Bryan Professor in the Departments of Medicine and Molecular Microbiology and co-director of the Division of Infectious Diseases in the School of Medicine, has used the FluoroDOT assay in her laboratory. .

“When Mycobacterium tuberculosis infects immune cells, those cells respond by secreting important immune proteins called cytokines,” Phillips said. “But not all cells respond to infection in the same way. The Fluorodot assay allowed us to see how individual cells in a population respond to infection — to see which cells are secreting which and which In the direction. It was not possible with the old technology.”


Washington University in St. Louis

Journal Reference:

Seth, A., and others. (2022) High-resolution imaging of protein secretion at the single-cell level using the plasmon-enhanced FluoroDot assay. Cell Report Methods. doi.org/10.1016/j.crmeth.2022.100267.

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