“Simple yet powerful”: See cell secretion like never before

We recently saw the stunning images of distant galaxies revealed by the James Webb Telescope, previously visible only as blurry dots. Researchers at Washington University in St. Louis have developed a novel method for visualizing the proteins secreted by cells with breathtaking resolution, making it the James Webb version of visualizing the protein secretion of individual cells.

The researchers, led by Srikanth Singamaneni, the Lilyan & E. Lisle Hughes Professor of Mechanical Engineering and Materials Science at the McKelvey School of Engineering, and Anushree Seth, a former postdoctoral researcher in Singamaneni’s lab, developed the FluoroDOT test, which they introduced Paper in the journal on August 5, 2022 Cell Reports methods. The highly sensitive assay is able to see and measure proteins secreted by a single cell in approximately 30 minutes.

Working with researchers from Washington University School of Medicine and other universities, they found that the FluoroDOT assay is versatile, inexpensive, adaptable to any laboratory setting, and has the potential to provide a more comprehensive look at these proteins than the widely used one existing assays. Biomedical researchers look to these secreted proteins for information on cell-to-cell communication, cell signaling, activation, and inflammation, among other things, but existing methods are limited in their sensitivity and can take up to 24 hours to process.

What sets the FluoroDOT assay apart from existing assays is the use of a plasmon fluor, a plasmon-enhanced nanolabel developed in Singamaneni’s lab that is 16,000 times brighter than traditional fluorescent labels and has a signal-to-noise ratio nearly 30 times higher.

“Plasmonic fluores are composed of metal nanoparticles that act as an antenna to attract the light and enhance the fluorescence emission from molecular fluorophores, making them ultra-bright nanoparticles,” Singamaneni said.

This ultra-bright emission from Plasmon-Fluor allows the user to see extremely small amounts of secreted protein, which is not possible in existing assays, and digitally measure the high-resolution signals using the number of particles or dot pattern per cluster. or Spot, with a custom algorithm. Also, no special equipment is required. Singamaneni and his co-workers first published their work with plasmon fluorine in Nature Biomedical Engineering in 2020.

The patent-pending plasmonic fluorine technology is licensed to Auragent Bioscience LLC from the Office of Technology Management at Washington University in St. Louis.

“Using a simple fluorescence microscope, we can simultaneously image a cell along with the spatial distribution of the proteins secreted around it,” said Seth, who worked on this project as a postdoctoral fellow in Singamaneni’s lab and remains a senior scientist (cellular applications ) for Auagent Bioscience. “We have seen interesting secretion patterns for different cell types. This assay also allows the simultaneous visualization of two types of proteins from single cells. When multiple cells are exposed to the same stimuli, we can distinguish the cells that secrete two proteins at the same time from those that secrete only one protein or no protein at all.”

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

One of the collaborators and co-authors, Jennifer A. Philips, MD, Ph.D., Theodore and Bertha Bryan Professor in the Departments of Medicine and Molecular Microbiology and Co-Director of the Department of Infectious Diseases in the School of Medicine, created the FluoroDOT -Assay used in her lab.

“When Mycobacterium tuberculosis infects immune cells, those cells respond by secreting important immune proteins called cytokines,” Philips said. “But not all cells respond to infection in the same way. Using the FluoroDOT assay, we were able to see how individual cells in a population respond to infection – to see which cells are secreting and in which direction. This was not possible with the older technology.”