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Quantification and Size-profiling of Extracellular Vesicles Using Tunable Resistive Pulse Sensing
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A liposome-based size calibration method for measuring microvesicles by flow cytometry.

J B Simonsen1

  • 1Department of Chemistry and Nano-Science Center, University of Copenhagen, Copenhagen, Denmark.

Journal of Thrombosis and Haemostasis : JTH
|October 29, 2015
PubMed
Summary

Accurate microvesicle sizing by flow cytometry requires a gold standard. Fluorescent liposomes serve as superior size calibrators compared to polystyrene beads, enhancing microvesicle size assessment.

Keywords:
calibrationcell-derived microparticlesflow cytometryliposomesreference standards

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Area of Science:

  • Extracellular vesicle research
  • Flow cytometry applications
  • Nanoparticle characterization

Background:

  • Accurate sizing of microvesicles is crucial for understanding their biological roles.
  • Existing methods for microvesicle size determination by flow cytometry lack a standardized gold standard.
  • The need for reliable calibrators in flow cytometry-based vesicle analysis is increasingly recognized.

Purpose of the Study:

  • To establish a more accurate method for determining microvesicle sizes using flow cytometry.
  • To evaluate the suitability of fluorescently labeled liposomes as size calibrators.
  • To compare the performance of liposome-based calibrators against traditional polystyrene beads.

Main Methods:

  • Preparation of fluorescently labeled liposomes with defined sizes (200-1000 nm) via extrusion.
  • Measurement of side scattering (SSC) signals from liposomes and microvesicles using flow cytometry.
  • Correlation analysis between liposome size, fluorescence intensity, and SSC signals.

Main Results:

  • Fluorescent liposomes demonstrated a reliable correlation between SSC and vesicle size.
  • Polystyrene beads showed significant inaccuracies, with 110 nm beads correlating to ~400 nm vesicles.
  • The SSC-based size assessment method exhibits a resolution limit of approximately 60-200 nm.

Conclusions:

  • Fluorescent liposomes offer a more accurate size calibration method for microvesicles in flow cytometry.
  • Liposome-based calibration improves the reliability of microvesicle size estimation compared to polystyrene beads.
  • The study highlights the limitations and potential improvements for flow cytometry-based vesicle sizing.