Exosome staining you can trust

ExoBrite™ EV Membrane Staining Kits were designed to overcome some of the challenges of exosome detection. ExoBrite™ stains bind to molecules in the exosome membrane providing bright, specific staining of isolated EVs by flow cytometry. A key advantage of ExoBrite™ stains is that, unlike many other dyes, they show little to no background aggregates of a similar size as exosomes or EVs. Also, unlike most membrane dyes, ExoBrite™ stains do not bind non-specifically to polystyrene beads, meaning that they can be used to stain bead-bound exosomes.

ExoBrite™ EV Membrane Stains have been validated in flow cytometry for their ability to stain exosomes and EVs derived from several different cultured cell lines, and isolated by several different methods, including size exclusion chromatography (SEC), polyethylene glycol (PEG) precipitation, and magnetic bead immunoprecipitation (IP).

ExoBrite™ EV Membrane Stain advantages:

Designed for exosome detection by flow cytometry
Bright fluorescence and low background for excellent signal-to-noise
Compatible with antibody co-staining (unlike CellBrite® Fix or MemBrite® Fix)
Stain purified or bead-bound exosomes
Available in 4 colors for flexible experimental design

SEC-purified MCF-7-derived exosomes were stained with ExoBrite™ EV Membrane Stains.

Less background and better coverage over other exosome/EV stains

ExoBrite™ EV Membrane Stains have less background and more complete exosome staining than other classic dye or competitor dyes. Exosomes were purified from MCF-7 cell supernatant using size exclusion chromatography (SEC). The purified exosomes were stained in PBS with the indicated dyes (top row). The exosome population was gated, with the number showing the percentage of particles falling within the exosome gate. Each dye was also added to filtered PBS (bottom row), to look for dye aggregation and non-specific background. Red arrows indicate these dye aggregates. Lipophilic dyes like DiO show a high number of particles of similar size to exosomes, making them unsuited for exosome staining. CellMask™ also shows an unacceptable amount of dye aggregation falling within the exosome gate. The ExoFlow-ONE™ and ExoGlow™ dyes form aggregates that can mostly be gated away from the true exosomes, but they also show less-complete coverage of exosomes than ExoBrite™ membrane stains (Click to enlarge).

Product	Ex/Em	Detection channels	Size	Catalog Number
ExoBrite™ 410/450 EV Membrane Staining Kit	416/452 nm	Pacific Blue™	100 Labelings	30111-T
			500 Labelings	30111
ExoBrite™ 490/515 EV Membrane Staining Kit	490/516 nm	FITC	100 Labelings	30112-T
			500 Labelings	30112
ExoBrite™ 560/585 EV Membrane Staining Kit	562/584 nm	PE, Cy®3	100 Labelings	30113-T
			500 Labelings	30113
ExoBrite™ 640/660 EV Membrane Staining Kit	642/663 nm	APC	100 Labelings	30114-T
			500 Labelings	30114

Purified exosomes stained with ExoBrite™ EV membrane stains

SEC-purified MCF-7-derived exosomes were stained with ExoBrite™ 490/515 EV Membrane Stain (right). Specific staining was seen, compared with the same stain in buffer (left). Exosomes were detected on a CytoFLEX LX flow cytometer in the FITC channel.

SEC-purified MCF-7-derived exosomes were stained with ExoBrite™ 560/585 EV Membrane Stain (right). Specific staining was seen, compared with the same stain in buffer (left). Exosomes were detected on a CytoFLEX LX flow cytometer in the PE channel.

Bead-bound EVs stained with ExoBrite™ EV membrane stains

Flow cytometry of bead-bound exosomes derived from MCF-7 cells. Unstained exosomes (gray) or stained with 10X ExoBrite™ 490/515 (green). Exosomes were detected on a CytoFLEX LX flow cytometer in the FITC channel.

Flow cytometry of bead-bound exosomes derived from MCF-7 cells. Unstained exosomes (gray) or stained with 10X ExoBrite™ 560/585 (orange). Exosomes were detected on a CytoFLEX LX flow cytometer in the R-PE channel.

Flow cytometry of bead-bound exosomes derived from MCF-7 cells. Unstained exosomes (gray) or co-stained with 10X ExoBrite™ 490/515 and CD81-CF®568 (pink). Exosomes were detected on a CytoFLEX LX flow cytometer in the FITC and R-PE channels.

Flow cytometry of bead-bound exosomes derived from MCF-7 cells. Unstained exosomes (gray) or co-stained with 10X ExoBrite™ 560/585 and CD9(HI9a)-CF®488A (pink). Exosomes were detected on a CytoFLEX LX flow cytometer in the FITC and R-PE channels.

ExoBrite™ EV membrane stain/antibody co-staining

One advantage of ExoBrite™ EV Membrane Stains is the ability to co-stain with antibodies, for exosome protein profiling studies. Co-staining can be performed concurrently or sequentially, and can be performed on purified or bead-bound exosomes. ExoBrite™ EV Membrane Stains are general exosome stains that should label all exosomes in a sample,* allowing antibodies to be used to quantify how many exosomes express the protein of interest. For example, in the exosomes that we tested (MCF-7-derived), the tetraspanin CD9 is highly and ubiquitously expressed.** We have found that when we performed co-staining with ExoBrite™ 560/585 EV Membrane Stain and CD9(H19a)-CF®488A, they show near-complete overlap of their positive populations (see Figure, right).

Tips for optimal ExoBrite™/antibody co-staining of purified exosomes:

Stain purified exosomes in 1 mL with 10X ExoBrite™ (ExoBrite™ staining is somewhat reduced during simultaneous co-staining so 1X is not recommended)
Try starting with at least 0.1 ug/mL antibody

*In the exosomes that we have tested, ExoBrite™ EV Membrane Stains label all exosomes. There may be exosome types with different staining levels.

**Expression levels of tetraspanin proteins (CD9, CD61, & CD81) and other proteins differ greatly between cell types and exosome sources.

SEC-purified MCF-7-derived exosomes were stained simultaneously with 1 mL 10X ExoBrite™ 560/585 EV Membrane Stain and 0.1 ug/mL CD9(H19a)-CF®488A. When gated on ExoBrite™ 560/585-positive particles, ~95% were also positive for CD9 (top row). Likewise, when gated on CD9-positive particles, ~ 95% were also positive for ExoBrite™ 560/585 (bottom row)

CellMask is a trademark of Thermo Fisher Scientific; ExoFlow-ONE and ExoGlow are trademarks of System Biosciences.