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.
The benefits of using flow cytometry include:
- Single particle analysis—traditional methods of EV analysis are limited in the capacity to analyze individual cells and often are time-consuming or expensive. Flow cytometry is capable of analyzing thousands of cells per second, improving statistics, and allowing the quantification of unique and rare cell types.
- Accurate particle counting—the Attune Flow Cytometers have significant advantages in both speed and accuracy for absolute counting methods.
- Ability to quickly multiplex with reagents and antibodies—get more information by simultaneous analysis with fluorescent labels such as surface proteins, membrane lipids, cellular esterase and antibodies targeting cell surface and intracellular markers.
Learn about the Attune Flow Cytometers and which antibodies and reagents identify and characterize EVs.
Attune Flow Cytometers minimize coincidences to provide more accurate single-particle analysis
EVs often require significant sample dilution to ensure single-particle detection. Higher particle concentrations in a sample results in higher coincidences, also known as the swarm effect (Figure 1). Attune Flow Cytometers are equipped with acoustic-assisted hydrodynamic focusing, which allows analyzing highly diluted samples quickly and without increasing the number of coincidences (Figure 2). We recommend that you add the 488/10 side scatter filter along with ultra-filtering of the focusing fluid to decrease background noise and enable discrimination of 100 nm particles.
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.
Antibodies and reagents to characterize EVs
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)
Detect your EV population from debris with these simple tips
Controls
Controls are important to correctly identify your population of EVs from contamination and debris. Remember to include these controls:
- Buffer only
- Buffer + all reagents (no EVs)
- Buffer + EVs only (no stain)
- Sample dilutions of EVs in buffer to ensure single particle detection
- Particles for instrument characterization
- If using multiple flow cytometry antibodies, have FMO controls
- 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.
Sample preparation
You may also employ Dynabeads magnetic beads as an alternative method for EV isolation and flow cytometry detection.
Free webinar to learn exosome analysis workflow using flow cytometry
Title: Analysis of Surface Antigens on Exosomes Using the Attune NxT Flow Cytometer
Presenter: Steve McClellan, BS, MT, SCYM (ASCP)CM Manager, Basic & Translational Research Operations Chief, Flow Cytometry Core Laboratory, Mitchell Cancer Institute University of South Alabama
Webinar highlights:
- Exosome isolation methods
- Best practices for flow cytometry exosome analysis
- Surface markers analysis for exosomes
- Importance of exosome research
References
- Lötvall J, Hill AF, Hochberg F, et al. Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. J Extracell Vesicles. 2014;3:26913. Published 2014 Dec 22.
- Witwer KW, Buzás EI, Bemis LT, et al. Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J Extracell Vesicles. 2013;2:10.3402/jev.v2i0.20360. Published 2013 May 27.
Resources
Flow Cytometry Learning Center—Access flow cytometry educational resources for better experiment planning and execution.
Flow Cytometry Panel Builder—Design your flow cytometry panel with this online tool for a simplified, customizable experience to fit your needs.
Support
Flow Cytometry Support Center—Find technical support recommendations for your flow cytometry workflows, including tips for experimental setup and in-depth troubleshooting help.