Exosomes become visible with flow cytometry

Although they were discovered nearly 30 years ago, exosomes have only recently moved into the research spotlight. Now, biomedical researchers are examining the contents and characteristics of exosomes as possible biomarkers for cancer and other diseases because they can be sampled non-invasively. Biologists are uncovering a diversity of functional changes elicited in cells upon taking up exosomes via membrane fusion or ligand-receptor interactions.

Previously believed to be no more than garbage disposal systems of the cell, evidence is rapidly accumulating to the role of exosomes as messengers conveying information between cells. As small membrane vesicles released by endocytosis, exosomes carry proteins, lipids, miRNA and mRNA specific to the type of cell from which they originate, be it T cells, B cells, dendritic cells, platelets, neurons, or epithelial cells. They transport this payload as they travel in biological fluids to distant tissues.

Due to their small size (≤150 nm), capturing and examining exosomes has proven challenging. Detection of single exosomes is very difficult via flow cytometry, but it is possible to gain insights about a population of exosomes. Proteins on the surface of exosomes can be stained with fluorochrome-conjugated antibodies to characterize populations originating from a particular cell type.

Clever solution, straightforward protocol

The exosome population to be characterized can be isolated from cell culture supernatant or plasma via differential ultracentrifugation and microfiltration. A sample containing at least 500 ng exosome protein is then incubated with a specific antibody conjugate (e.g., CD63-APC). Multiple stainings are possible, as long as the dyes used do not overlap. Also make sure to have:

1. a buffer control to eliminate background noise
2. a sample of unstained exosome to check for autofluorescence, and
3. an isotype control to determine non-specific binding of the antibody conjugate

Then run your samples on the flow cytometer and use gating to isolate the data from the stained exosome clusters.

Detailed Protocol- Characterization of exosome populations using the MACSQuant® Analyzer