Extracellular Vesicle Detection with Flow Cytometry

Rapidly detect and quantitate single particles

Extracellular vesicles (EVs) including exosomes are being actively researched. EVs are involved with several biological processes including (1) cell-to-cell signaling; (2) transfer proteins, lipids, and nucleic acids; and (3) markers of disease. The small size of EVs, their heterogeneity, and low refractive index make them difficult to identify. However, flow cytometry can be used to analyze single particles and provide both cell counting and phenotyping of EV properties.

Figure 1. Highly concentrated samples produce ‘swarm’ signals with multiple vesicles detected as a single event. Minimize the high the high coincidence with serial sample dilutions.

Figure 2. Attune Flow Cytometers use acoustic-assisted hydrodynamic focusing to run very dilute samples with lower coincident events.

Specific targets

Antigen expression depends on the cell of origin. These markers should be used in combination, with specific markers for the cell of origin or other reagents.

• CD9, CD63, CD81 (cell-surface markers) are mainly used for exosomes.
• Brighter fluorochromes including PE and APC are good choices for detection since the number of molecules are low per EV. Tandems are not advised.

Membrane dyes

Fluorescent dyes can be used to uniformly label a population of EVs from cell culture. These stain lipids and cell membranes which is useful for EV gating and detection.

• FM lipophilic styryl dyes: FM 1-43
• Long-chain lipophilic carbocyanine dyes: DiI, CM-DiI (fixable), DiO, and DiD or Vybrant Multicolor Cell Labeling Kit
• Di-8-ANNEPS

Intracellular dyes

Intracellular proteins and DNA can be labeled with bright dyes. RNA staining is more challenging. We offer several dyes in a wide range of colors to help detect EV populations.

• CellTrace and CFSE dyes (intravesicular proteins)
• Hoechst 33342 (DNA stain)

Controls

Controls are important to correctly identify your population of EVs from contamination and debris. Remember to include these controls:

1. Buffer only
2. Buffer + all reagents (no EVs)
3. Buffer + EVs only (no stain)
4. Sample dilutions of EVs in buffer to ensure single particle detection
5. Particles for instrument characterization
6. If using multiple flow cytometry antibodies, have FMO controls
7. Negative control with Triton™ X-100 treatment. With Triton™ X-100 treatment, the EV membrane should be disturbed and any putative EV signal should be lost. Otherwise, the observed signal could be due to artifacts.

Prevention of contamination

Filter all fluids and wash pipet tips with filtered distilled water to minimize contamination of debris and non-sample EVs. We recommend that you use 0.1 μm or smaller filters. A 500 ml bottle will require 12–24 hours of vacuum to filter all fluids.