Antibody titrations are critical for microflow cytometric analysis of extracellular vesicles

Optimization of flow cytometry assays for extracellular vesicles (EVs) often fail to include appropriate reagent titrations – the most critically antibody titration is either not performed or is incomplete. Using nonoptimal antibody concentration is one of the main sources of error leading to a lack...

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Bibliographic Details
Published in:Cytometry. Part A 2023-08, Vol.103 (8), p.670-683
Main Authors: Pink, Desmond, Basu, Arghya, Wong, Michael, Pham, Diana, Valencia, Juliana, Triana, Vivian, Beatty, Perrin H., Rieger, Aja M., Lewis, John D.
Format: Article
Language:English
Subjects:
Antibodies
antibody titration
Antigens
Control equipment
EVs
Extracellular vesicles
Flow cytometry
microflow cytometry
Optimization
PDPs
plasma
Platelets
platelet‐derived particles
Reagents
Separation Index
Stain Index
Titration
Vesicles
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Summary:Optimization of flow cytometry assays for extracellular vesicles (EVs) often fail to include appropriate reagent titrations – the most critically antibody titration is either not performed or is incomplete. Using nonoptimal antibody concentration is one of the main sources of error leading to a lack of reproducible data. Antibody titration for the analysis of antigens on the surface of EVs is challenging for a variety of technical reasons. Using platelets as surrogates for cells and platelet‐derived particles as surrogates for EV populations, we demonstrate our process for antibody titration, highlighting some of the key analysis parameters that may confound and surprise new researchers moving into the field of EV research. Additional care must be exercised to ensure instrument and reagent controls are utilized appropriately. Complete graphical analysis of positive and negative signal intensities, concentration, and separation or stain index data is highly beneficial when paired with visual analysis of the cytometry data. Using analytical flow cytometry procedures optimized for cells for EV analysis can lead to misleading and nonreproducible results.
ISSN:1552-4922
1552-4930
DOI:10.1002/cyto.a.24733