Exosomes are membrane-bound vesicles actively secreted by cells, typically 30–150 nm in diameter. They contain various biomolecules derived from host cells, such as proteins, DNA, coding and non-coding RNAs, and lipids. Exosomes are closely associated with tumor formation, growth, metastasis, and disease progression. As such, they have become a research hotspot in tumor diagnosis and therapy.
Labeled exosomes are commonly used to study uptake by recipient cells and for in vivo biodistribution imaging in small animals. So, what are the key tips to ensure high-efficiency fluorescent labeling of exosomes?
Q1: What fluorescent dyes can be used for exosome labeling?
Lipophilic membrane dyes are commonly used for convenient exosome labeling. These dyes work by embedding their long aliphatic tails into the phospholipid bilayer of the exosome membrane, enabling strong fluorescent signals.
Commonly used lipophilic fluorescent probes include:
PKH67 (green; Ex/Em = 490/502 nm)
PKH26 (red; Ex/Em = 551/567 nm)
DiO (green; Ex/Em = 484/501 nm)
DiI (red; Ex/Em = 549/565 nm)
DiD (far-red; Ex/Em = 644/663 nm)
DiR (near-infrared; Ex/Em = 750/780 nm)
Choosing the right dye is essential for successful labeling. DiO and DiI are suitable for co-culture experiments with cells, while DiR is better suited for in vivo imaging of exosomes.
Q2: How to determine the appropriate labeling concentration?
While DiO and DiI are typically used at 1–5 μM for cell membrane labeling, this concentration is relatively low for exosome labeling. To ensure high labeling efficiency, it is recommended to use a 100 μM concentration for DiO, DiI, and DiR. This concentration promotes efficient labeling, is non-toxic to exosomes, and is easy to remove due to the dyes’ small molecular size.
Q3: Is fixation required for exosome labeling?
No fixation is needed. Similar to live-cell labeling, lipophilic dyes like DiO can be used directly on exosomes without the need for fixation or permeabilization.
Q4: What is the recommended dye volume relative to exosome quantity?
Once the dye is prepared as a 100 μM working solution, follow this guideline based on exosomal protein content:
|
Exosomal Protein Amount |
Volume of Dye Working Solution |
|
10–200 μg |
50 μL |
|
200–500 μg |
100 μL |
|
500–1000 μg |
200 μL |
Q5: What are the incubation conditions for labeling?
Incubate the exosomes with dye at room temperature or 37°C for 30 minutes.
Q6: How to remove free/unbound dye after labeling?
After incubation, add PBS at least 50 times the labeling volume for washing. Use one of the following methods to remove excess dye:
Ultracentrifugation: Centrifuge at 120,000 × g for 1 hour.
Ultrafiltration: Use a 100 kDa or 10 kDa ultrafiltration tube. The 100 kDa filter has pore sizes around 10 nm, allowing free dye to pass through while retaining labeled exosomes. After centrifugation, invert the inner tube into a new centrifuge tube and centrifuge again to collect the labeled exosomes.
Q7: How to assess labeling success?
Labeled exosomes can be detected using fluorescence microscopy or in vivo imaging systems. Successfully labeled exosomes exhibit strong fluorescence, while unsuccessful labeling yields little to no signal (as illustrated below).
Q8: How to store labeled exosomes?
If not used immediately, labeled exosomes can be stored at –80°C. For short-term storage, 4°C is acceptable.
Conclusion
Exosome labeling is relatively straightforward. The key determinants of labeling efficiency are the choice of dye and its working concentration. Selecting appropriate dyes and using optimal concentrations can significantly improve labeling outcomes. We hope these FAQs will assist junior researchers in successfully labeling exosomes and resolving related challenges. Using the right tools and methods will make your work more efficient and effective.
References
[1] Li Ning. Research on Exosome Membrane Fluorescence Analysis and Imaging Methods [D]. Dalian University of Technology, 2020. DOI: 10.26991/d.cnki.gdllu.2020.004153.
[2] Ma W, Zhang X, Liu Y, et al. Polydopamine Decorated Microneedles with Fe-MSC-Derived Nanovesicles Encapsulation for Wound Healing. Adv Sci (Weinh). 2022;9(13):e2103317. doi:10.1002/advs.202103317
[3] Lobb R. J., Becker M., Wen S. W., et al. Optimized Exosome Isolation Protocol for Cell Culture Supernatant and Human Plasma. J Extracell Vesicles. 2015;4(1):27031.
Product Overview
|
Product Name |
Specification |
Cat No. |
|
HieffTM Quick exosome isolation kit（for Serum/Plasma） |
30 ml |
41202ES30 |
|
HieffTM exosome tracker kit(for green fluorescence) |
20 µl |
40781ES20 |
|
DiO (DiOC18(3)) |
10 mg/25 mg |
40725ES10/25 |
|
DiI (DiIC18(3)) |
10 mg |
40726ES10 |
|
DiD Perchlorate (DiIC18(5)) |
25 mg |
40758ES25 |
|
DiR Iodide (DiIC18(7)) |
25 mg |
40757ES25 |