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What is the best way to store exosomes?
For short-term storage, exosomes can be kept at 4 °C for up to one week. For long-term storage, they can be stored at −20 °C or −80 °C. When storing exosomes long-term, it is important to consider whether multiple thawing events will be needed. If repeated use is required, we recommend aliquoting the exosome solution into multiple tubes so that each tube undergoes only a single freeze–thaw cycle. Multiple freeze–thaw cycles have been found to damage exosomes and reduce their quantity.
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What are the current methods for isolating exosomes?
Isolating exosomes from cell culture media or bodily fluids is a complex and challenging process. It typically involves ultracentrifugation combined with sucrose density gradient or sucrose cushion centrifugation, allowing low-density exosomes to be separated from other vesicles and particles. These procedures can take up to 30 hours and require an ultracentrifuge as well as extensive training. Despite these drawbacks, this remains the most commonly used method for exosome isolation.
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How can the quantity of exosomes be quantified?
Nanoparticle tracking analysis (NTA) is currently one of the most commonly used methods to determine the average particle size and concentration in solution. However, this technique cannot distinguish vesicles from protein aggregates of similar size. Therefore, we recommend combining NTA with microscopy techniques to verify the presence of membrane-bound vesicles. We place great emphasis on consistent cell culture and exosome collection conditions to ensure comparable exosome yields between preparations, which we consider more important than simply estimating the total protein concentration of the preparation.
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What do exosomes contain?
Depending on their origin and function, exosomes can contain a variety of components, such as immune factors, growth factors, microRNAs, and mRNAs. The applications of exosomes depend on the specific substances they carry.
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How are exosomes defined?
At present, the definition of exosomes is relatively complex, and there is no absolute consensus in the field. Exosomes are commonly defined as vesicles with a density of approximately 1.13–1.19 g/mL that float in a sucrose gradient during ultracentrifugation, with an expected size of 30–150 nm based on electron microscopy analysis. Exosomes can also be defined and identified by their surface protein markers, including tetraspanins (CD63, CD81, CD9) and other markers such as ALIX.
