Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Beforesharing sensitive information, make sure you’re on a federalgovernment site.

Https

The site is secure.
Thehttps://ensures that you are connecting to theofficial website and that any information you provide is encryptedand transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
.2020 Apr 27;8(5):98.
doi: 10.3390/biomedicines8050098.

Antibody-Free Labeling of Malaria-Derived Extracellular Vesicles Using Flow Cytometry

Elya Dekel 1, Paula Abou Karam 1, Yael Ohana-Daniel 1, Mirit Biton 1, Neta Regev-Rudzki 1, Ziv Porat 2
Affiliations

Antibody-Free Labeling of Malaria-Derived Extracellular Vesicles Using Flow Cytometry

Elya Dekel et al. Biomedicines. .

Abstract

Extracellular vesicles (EVs) are cell-derived membrane-bound structures that are believed to play a major role in intercellular communication by allowing cells to exchange proteins and genetic cargo between them. In particular, pathogens, such as the malaria parasitePlasmodium (P.) falciparum, utilize EVs to promote their growth and to alter their host's response. Thus, better characterization of these secreted organelles will enhance our understanding of the cellular processes that govern EVs' biology and pathological functions. Here we present a method that utilizes a high-end flow cytometer system to characterize small EVs, i.e., with a diameter less than 200 nm. Using this method, we could evaluate different parasite-derived EV populations according to their distinct cargo by using antibody-free labeling. It further allows to closely monitor a sub-population of vesicles carrying parasitic DNA cargo. This ability paves the way to conducting a more 'educated' analysis of the various EV cargo components.

Keywords:Plasmodium falciparum; extracellular vesicles; flow cytometry; malaria.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Detection of nanoparticles using the ZE5 analyzer and NTA analysis. (A) Polystyrene beads of 200 nm (top row, middle panel) and 100 nm (top row, right panel) in diameter were gated according to FSC 405/10 and SSC 488/10 light scattering. The analysis was visualized with a density plot and the gating was performed according to the main population. Rhodamine-PE large unilamellar vesicles (LUVs) were detected according to FSC 405/10 and the Rhodamine distribution. A PBS sample was used as control for undetected particles. (B) Nanoparticle tracking analysis (NTA) (Malvern Instruments Ltd., Nanosight NS300) was performed at 20 °C on Rho-LUVs and EVs to determine the concentration and size. Sample size distributions were calibrated in a liquid suspension (1:1000 dilution) by the analysis of Brownian motion via light scattering. The camera level was set to 13 and the gain to 1, laser 405 nm or 488 nm without filter.
Figure 2
Figure 2
Detection of different types and amounts ofPf-derived EV cargo using the ZE5 analyzer. (A)Pf-derived EVs were stained using three different dyes to visualize different types of cargo components: proteins with the CFSE marker (top row), nucleic acids with the HO marker (middle row), and lipids with the PKH26 marker (bottom row). As controls, solutions containing free dye or unstained EVs were used. (B) Detection of differentPf-derived EV concentrations was achieved by diluting the EVs with the same dye (PKH26 and HO) amount. On the left: high concentration (8 × 1011par/mL); in the center and on the right: lower concentrations of EVs.
Figure 2
Figure 2
Detection of different types and amounts ofPf-derived EV cargo using the ZE5 analyzer. (A)Pf-derived EVs were stained using three different dyes to visualize different types of cargo components: proteins with the CFSE marker (top row), nucleic acids with the HO marker (middle row), and lipids with the PKH26 marker (bottom row). As controls, solutions containing free dye or unstained EVs were used. (B) Detection of differentPf-derived EV concentrations was achieved by diluting the EVs with the same dye (PKH26 and HO) amount. On the left: high concentration (8 × 1011par/mL); in the center and on the right: lower concentrations of EVs.
Figure 3
Figure 3
Detection of different subpopulations ofPf-derived EVs using the ZE5 analyzer. (A) Top row: Mixed population ofPf-derived EVs (ring and trophozoite) stained with HO. Gating was done according to the unstained EVs. Bottom row: medium-derived ‘EVs’ were used as a control for absence of DNA content. (B)Pf-derived EVs from different parasite stages (ring and trophozoite) were stained with HO to visualize the DNA inside the EVs. Gating was done according to the unstained EV population.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Malda J., Boere J., van de Lest C.H.A., van Weeren P.R., Wauben M.H.M. Extracellular vesicles—New tool for joint repair and regeneration. Nat. Rev. Rheumatol. 2016;12:243. doi: 10.1038/nrrheum.2015.170.-DOI-PMC-PubMed
    1. Becker A., Thakur B.K., Weiss J.M., Kim H.S., Peinado H., Lyden D. Extracellular Vesicles in Cancer: Cell-to-Cell Mediators of Metastasis. Cancer Cell. 2016;30:836–848. doi: 10.1016/j.ccell.2016.10.009.-DOI-PMC-PubMed
    1. Van Dongen H.M., Masoumi N., Witwer K.W., Pegtel D.M. Extracellular Vesicles Exploit Viral Entry Routes for Cargo Delivery. Microbiol. Mol. Biol. Rev. 2016;80:369–386. doi: 10.1128/MMBR.00063-15.-DOI-PMC-PubMed
    1. Montaner S., Galiano A., Trelis M., Martin-Jaular L., del Portillo H.A., Bernal D., Marcilla A. The Role of Extracellular Vesicles in Modulating the Host Immune Response during Parasitic Infections. Front. Immunol. 2014;5:1–8. doi: 10.3389/fimmu.2014.00433.-DOI-PMC-PubMed
    1. Ridder K., Sevko A., Heide J., Dams M., Rupp A.K., Macas J., Starmann J., Tjwa M., Plate K.H., Sültmann H., et al. Extracellular vesicle-mediated transfer of functional RNA in the tumor microenvironment. Oncoimmunology. 2015;4:e1008371. doi: 10.1080/2162402X.2015.1008371.-DOI-PMC-PubMed

LinkOut - more resources