Detection and Quantification of Extracellular Vesicles via FACS: Membrane Labeling Matters!

Fanny Ender,Frank Gieseler,Piet Zamzow,Nikolas Von Bubnoff

doi:10.3390/ijms21010291

Abstract

The field of extracellular vesicle (EV) research is challenged by the lack of standardized protocols to identify and specifically distinguish between exosomes and ectosomes, which are released via exocytosis or plasma membrane shedding, respectively. Using sequential centrifugation, we separated EV subpopulations from supernatants of COLO 357 pancreas carcinoma cells based on size and mass. After 10,000× g centrifugation, we reconstituted high-speed (hs) EVs from the pellet, directly labeled them with the membrane dye carboxyfluorescein diacetate succinimidyl ester (CFSE), and performed flow cytometry based analysis. The aim was to optimize the conditions for EV labeling and detection and hence to obtain a maximum yield of intact hsEVs. We found that, for sufficient labeling of EVs, minimal temperature variations and short incubation times correlated with EV stability. Furthermore, threshold adjustment significantly improved the sensitivity of the flow cytometer for the detection of CFSE labeled hsEVs. When cells were CFSE labeled, we observed a transition of fluorescence onto EVs that were reconstituted from the pellet but not onto those that remained in the supernatant after hs centrifugation, suggesting the indirect labeling of EVs based on the way of biogenesis as a specific method for the distinction of exosomes and ectosomes. Protocol standardization is of major importance for the use of EVs as diagnostic markers in liquid biopsies.

Highlights

Before the development of high-resolution flow cytometers, small particles were excluded from the analysis of cellular preparations because they were seen as cellular debris or background noise
We showed before that high-speed centrifugation separates extracellular vesicle (EV) subpopulations based on size and density and on their coagulative capacity, since the amount of tissue factor (TF) activity was significantly higher on the surface of high-speed EVs after 10,000× g centrifugation when compared to smaller EVs that resided in the supernatant of malignant effusions from patients [7]
Scanning electron microscopic images of COLO 357 cell line-derived hsEVs show small, round structures that we identified as EVs (Figure 1C)

Summary

Introduction

Before the development of high-resolution flow cytometers, small particles were excluded from the analysis of cellular preparations because they were seen as cellular debris or background noise. The most promising approach to separate ectosomes from exosomes is a combination of different methods for EV purification (e.g., sequential centrifugation, magnetic beads) and characterization (e.g., high-resolution flow cytometry, nanoparticle tracking analysis (NTA), electron microscopy). Enabling multi-parameter single particle analysis of EVs, high-resolution flow cytometry is considered as the most promising technique for ideal EV analysis. A high-resolution flow cytometer typically differs from a conventional machine by having: (1) high powered lasers with a smaller focused beam spot size, (2) a stable, slow velocity core stream with a small diameter, (3) smaller fluorescence/side scatter collection optical apertures and/or higher sensitivity detectors, and (4) larger forward scatter obscuration bars and higher sensitivity detectors [8]

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Conclusion