A liposome-based size calibration method for measuring microvesicles by flow cytometry.
ESSENTIALS: A gold standard to determine the sizes of microvesicles by flow cytometry is needed. We used fluorescently labeled liposomes to estimate the size of microvesicles with flow cytometry. We suggest that liposomes are more accurate size calibrators than the commonly used polystyrene beads. The liposome-based size calibrators improve the size assessment of microvesicle made with flow cytometry.


BACKGROUND
During the past years, the need for a gold standard to determine the sizes of extracellular vesicles including microvesicles by flow cytometry has been emphasized.


METHODS
This work suggests that artificial vesicles can be used as calibrators to estimate the size of microvesicles from the side scattering (SSC) measured with flow cytometry. We prepared fluorescently labeled liposomes with different maximum sizes defined by the pore size (200, 400, 800, and 1000 nm) of the membrane used for the extrusion. The fluorescence strengths from the largest liposomes pertaining to each pore size enabled us to verify the correlation between the SSC from a liposome and the corresponding size.


CONCLUSIONS
This study indicates that artificial vesicles are more accurate size calibrators compared to the commonly used polystyrene calibrator beads illustrated by the SSC from 110 nm polystyrene beads corresponds to the scattering from ~400 nm vesicle-like particles. We also show that this method of size assessment based on SSC has a low resolution that is roughly estimated to be between 60 and 200 nm, dependent on the vesicle size.
Department of Chemistry and Nano-Science Center, University of Copenhagen, Copenhagen, Denmark.
J B
JB
Simonsen
D002138:Calibration; D055252:Cell-Derived Microparticles; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008081:Liposomes / Q000737:chemistry*; D008567:Membranes, Artificial; D016011:Normal Distribution; D010316:Particle Size; D011137:Polystyrenes / Q000737:chemistry; D015203:Reproducibility of Results; D012542:Scattering, Radiation
D008081:Liposomes; D008567:Membranes, Artificial; D011137:Polystyrenes
calibration; cell-derived microparticles; flow cytometry; liposomes; reference standards