Exosome & EV Staining
CellBrite® Cytoplasmic Membrane Dyes do not efficiently stain EVs, but CellBrite® Fix, MemBrite® Fix, CellBrite® Steady, and other stains have been used for this application. However, for optimal staining of exosome membranes we recommend our ExoBrite™ EV Membrane Stains. See our Exosome & EV Labeling Technology Page for more information.
For anyone looking to stain exosome membranes, our first suggestion will be ExoBrite™ EV Membrane Stains. The advantages of ExoBrite™ EV Membrane Stains are:
- Designed and formulated especially for exosome staining
- Very low background for excellent signal-to-noise
- Easily combine with antibodies for co-stain (unlike reactive dyes like CellBrite® Fix or MemBrite® Fix)
- Able to be used with bead-bound exosomes (unlike most other membrane or reactive dyes)
However, we have found that some other membrane and cell surface stains are also able to stain exosomes. None of the other stains has the same low background and high signal-to-noise as ExoBrite™ EV Membrane Stains, but they may have other properties (such as fixability) that are desirable for certain assays.
Membrane dyes for EV detection by flow cytometry
| Membrane Dye | Validated in flow with: | Staining Level | Notes |
|---|---|---|---|
| CellBrite® Steady | • PEG-enriched HeLa-derived EVs • SEC-purified MCF-7-derived exosomes1 |
Strong staining | • Fluorescent membrane probes • All 5 colors validated for exosome detection • Non-covalent labeling • Dyes bind non-specifically to magnetic beads |
| MemBrite® Fix 405/430 | • PEG-enriched HeLa-derived EVs • SEC-purified MCF-7-derived exosomes1 |
Strong staining | • Fluorescent membrane probe for the Pacific Blue channel • Reactive dye for covalent labeling • May not be suitable for antibody co-staining |
| CellBrite® Fix 488 & 555 | • SEC-purified MCF-7-derived exosomes1 | Moderate staining | • Fluorogenic membrane probes for the FITC and PE/Cy3 channels • Reactive dye for covalent labeling • May not be suitable for antibody co-staining • Used in publication as exosome stain2 |
| Di-8-ANEPPS | • SEC-purified MCF-7-derived exosomes1 | Moderate staining | • Red fluorescent membrane probe • Non-covalent labeling • Less soluble than other dyes • Used in publications as exosome stain3,4 |
2Cell Host & Microbe (2018) Aug 8;24(2):208-220.e8 doi: 10.1016/j.chom.2018.07.006
3Cytometry A. (2016) Feb;89(2):196-206 doi: 10.1002/cyto.a.22787
4Anal. Chem. (2021) 93, 14, 5897–5905 doi.org/10.1021/acs.analchem.1c00253
Yes, exosomes and EVs can be stained simultaneously with an ExoBrite™ EV Membrane Stain and a fluorescent antibody.
With purified exosomes, we have seen good results when exosomes were stained in 1 mL of 1X ExoBrite™ plus 1 ug fluorescent antibody.
With bead-bound exosomes we have found that sequential staining with first antibody and then ExoBrite™ EV Membrane Stain, with a wash step in between, gives the best results. When the staining is done simultaneously, the antibody signal is unaffected but the ExoBrite™ signal is reduced by ~ 25 %. This was observed when bead-bound exosomes were stained in 100 uL of 10X ExoBrite™ plus 1 ug fluorescent antibody; using different ratios of ExoBrite™ and antibody may give different results.
We have tested all of our tetraspanin (CD9, CD63, & CD81) antibody clones for their ability to stain exosomes purified by size exclusion chromatography (SEC) in flow cytometry. The clones that worked well are listed in a table on the “Exosome & EV Labeling” technology page.
https://biotium.com/technology/exosome-ev-labeling/#antibodies
We have tested all of our tetraspanin (CD9, CD63, & CD81) and Ep-CAM antibody clones for their ability to stain exosomes bound to CD63-capture magnetic beads in flow cytometry. The clones that worked well are listed in a table on the “Exosome & EV Labeling” technology page.
https://biotium.com/technology/exosome-ev-labeling/#antibodies