Description
Extracellular vesicles (EVs) are heterogeneous, lipid-bound particles (50 – 1000 nm) naturally secreted by cells into the extracellular space. Exosomes, a subclass of EVs, are defined by their size (40 to 160 nm) and biogenesis (endosomal origin). EVs, which transport biomolecules like proteins, lipids and nucleic acids, are of interest to researchers for diagnostic and therapeutic purposes. The Incucyte®Exofluor EV Labeling Kit allows for real-time evaluation of cellular EV uptake and is validated for use with the Incucyte®Live-Cell Analysis System. The Incucyte®Exofluor EV Labeling Dye labels the plasma membrane of small EVs by covalently binding to proteins and amino acids. The irreversible binding to the EVs, along with the removal of free dye, reduces the risk of non-specific dye attachment to other plasma membranes.
The Incucyte®Exofluor EV Labeling Kit is designed to label EVs, such as exosomes, for investigation in live-cell assays using the Incucyte®Live-Cell Analysis System. The kit includes Incucyte®Exofluor EV Labeling Dye for labeling EVs, Vivaspin®concentrators for removing free dye, and Minisart®PES 0.22 µm filters for sterilization and removal of unbound aggregate dye. After processing, labeled EVs are added to cells, and uptake is measured by quantifying green fluorescence. The kit is compatible with EVs isolated using various methods and contains components for 10 labeling reactions. It is intended for use in the Incucyte®SX5, S3, or SX1 Live-Cell Analysis Systems that are configured with either a Green/Red or a Green/Orange/NIR Optical Module.
Recommended Use
We recommend that the Incucyte®Exofluor EV Labeling Dye is prepared at a 5 mM stock concentration using anhydrous DMSO (not supplied). The dye should then be diluted 1:10 with Phosphate Buffer Saline (Ca²+/Mg²+free) and used at a final concentration of 80 μM in a 30-minute labeling reaction. The volume of EVs needed for each reaction depends on the number of assay wells and the concentration of EVs per well. The recommended range of EVs is 1 – 4 μg of protein/well or 5 x 10⁷ – 1 x 10⁹ particles/well – these ranges have been validated via testing of a wide range of exosome extraction methods, however some optimization may be required. Vivaspin®2 columns are used to remove unlabeled dye from the labeled EVs, with a maximum load of 40 μg protein or 100 μL labeled EV sample. Exceeding these limits may cause membrane clogging or increased background fluorescence. After removing the free dye, a Minisart®PES 0.22 µm syringe filter is used for sterilization and removal of unbound aggregates.
