Precise analysis of single small extracellular vesicles using flow cytometry.
Sci Rep
; 14(1): 7465, 2024 03 29.
Article
in En
| MEDLINE
| ID: mdl-38553534
ABSTRACT
Methods that enable specific and sensitive quantification of small extracellular vesicles (sEVs) using flow cytometry are still under development. Aggregation or adsorption of antibodies causes sub-nano sized particles or non-specific binding and largely affects the results of flow cytometric analysis of single sEVs. Comparison of control IgG and target-specific IgG is inappropriate because they have different characters. Here, we evaluate four preparation methods for flow cytometry, including ultracentrifugation, density gradient centrifugation, size exclusion chromatography (SEC), and the TIM4-affinity method by using tetraspanin-deficient sEVs. The ultracentrifugation or density gradient centrifugation preparation method has large false-positive rates for tetraspanin staining. Conversely, preparation methods using SEC or the TIM4-affinity method show specific detection of single sEVs, which elucidate the roles of sEV biogenesis regulators in the generation of sEV subpopulations. The methods are also useful for the detection of rare disease-related markers, such as PD-L1. Flow cytometric analysis using SEC or the TIM4-affinity method could accelerate research into sEV biogenesis and the development of sEV-based diagnostics and therapies.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Extracellular Vesicles
Language:
En
Journal:
Sci Rep
Year:
2024
Document type:
Article
Affiliation country:
Japan