Characterization of microvesicles released from human red blood cells

Duc Bach Nguyen, Thi Bich Thuy Ly, Mauro Carlos Wesseling, Marius Hittinger, Afra Torge, Andrew Devitt, Yvonne Perrie, Ingolf Bernhardt

Research output: Contribution to journalArticle

36 Citations (Scopus)

Abstract

Extracellular vesicles (EVs) are spherical fragments of cell membrane released from various cell types under physiological as well as pathological conditions. Based on their size and origin, EVs are classified as exosome, microvesicles (MVs) and apoptotic bodies. Recently, the release of MVs from human red blood cells (RBCs) under different conditions has been reported. MVs are released by outward budding and fission of the plasma membrane. However, the outward budding process itself, the release of MVs and the physical properties of these MVs have not been well investigated. The aim of this study is to investigate the formation process, isolation and characterization of MVs released from RBCs under conditions of stimulating Ca2+ uptake and activation of protein kinase C. Experiments were performed based on single cell fluorescence imaging, fluorescence activated cell sorter/flow cytometer (FACS), scanning electron microscopy (SEM), atomic force microscopy (AFM) and dynamic light scattering (DLS). The released MVs were collected by differential centrifugation and characterized in both their size and zeta potential. Treatment of RBCs with 4-bromo-A23187 (positive control), lysophosphatidic acid (LPA), or phorbol-12 myristate-13 acetate (PMA) in the presence of 2 mM extracellular Ca2+ led to an alteration of cell volume and cell morphology. In stimulated RBCs, exposure of phosphatidylserine (PS) and formation of MVs were observed by using annexin V-FITC. The shedding of MVs was also observed in the case of PMA treatment in the absence of Ca2+, especially under the transmitted bright field illumination. By using SEM, AFM and DLS the morphology and size of stimulated RBCs, MVs were characterized. The sizes of the two populations of MVs were 205.8 ± 51.4 nm and 125.6 ± 31.4 nm, respectively. Adhesion of stimulated RBCs and MVs was observed. The zeta potential of MVs was determined in the range from - 40 mV to - 10 mV depended on the solutions and buffers used. An increase of intracellular Ca2+ or an activation of protein kinase C leads to the formation and release of MVs in human RBCs.
LanguageEnglish
Pages1085-1099
Number of pages15
JournalCellular Physiology and Biochemistry
Volume38
Issue number3
DOIs
Publication statusPublished - 4 Mar 2016

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Erythrocytes
Atomic Force Microscopy
Electron Scanning Microscopy
Protein Kinase C
Cell-Derived Microparticles
Acetates
Cell Membrane
Exosomes
Fluorescein-5-isothiocyanate
Annexin A5
Optical Imaging
Phosphatidylserines
Calcimycin
Population Density
Lighting
Centrifugation
Cell Size
Buffers
Fluorescence
Extracellular Vesicles

Keywords

  • red blood cells
  • microvesicles
  • phosphatidylserine
  • fluorescence imaging
  • cell adhesion

Cite this

Nguyen, D. B., Thuy Ly, T. B., Wesseling, M. C., Hittinger, M., Torge, A., Devitt, A., ... Bernhardt, I. (2016). Characterization of microvesicles released from human red blood cells. Cellular Physiology and Biochemistry, 38(3), 1085-1099. https://doi.org/10.1159/000443059
Nguyen, Duc Bach ; Thuy Ly, Thi Bich ; Wesseling, Mauro Carlos ; Hittinger, Marius ; Torge, Afra ; Devitt, Andrew ; Perrie, Yvonne ; Bernhardt, Ingolf. / Characterization of microvesicles released from human red blood cells. In: Cellular Physiology and Biochemistry. 2016 ; Vol. 38, No. 3. pp. 1085-1099.
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Nguyen, DB, Thuy Ly, TB, Wesseling, MC, Hittinger, M, Torge, A, Devitt, A, Perrie, Y & Bernhardt, I 2016, 'Characterization of microvesicles released from human red blood cells' Cellular Physiology and Biochemistry, vol. 38, no. 3, pp. 1085-1099. https://doi.org/10.1159/000443059

Characterization of microvesicles released from human red blood cells. / Nguyen, Duc Bach; Thuy Ly, Thi Bich; Wesseling, Mauro Carlos; Hittinger, Marius; Torge, Afra; Devitt, Andrew; Perrie, Yvonne; Bernhardt, Ingolf.

In: Cellular Physiology and Biochemistry, Vol. 38, No. 3, 04.03.2016, p. 1085-1099.

Research output: Contribution to journalArticle

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T1 - Characterization of microvesicles released from human red blood cells

AU - Nguyen, Duc Bach

AU - Thuy Ly, Thi Bich

AU - Wesseling, Mauro Carlos

AU - Hittinger, Marius

AU - Torge, Afra

AU - Devitt, Andrew

AU - Perrie, Yvonne

AU - Bernhardt, Ingolf

N1 - © 2016 The Author(s) Published by S. Karger AG, Basel.

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AB - Extracellular vesicles (EVs) are spherical fragments of cell membrane released from various cell types under physiological as well as pathological conditions. Based on their size and origin, EVs are classified as exosome, microvesicles (MVs) and apoptotic bodies. Recently, the release of MVs from human red blood cells (RBCs) under different conditions has been reported. MVs are released by outward budding and fission of the plasma membrane. However, the outward budding process itself, the release of MVs and the physical properties of these MVs have not been well investigated. The aim of this study is to investigate the formation process, isolation and characterization of MVs released from RBCs under conditions of stimulating Ca2+ uptake and activation of protein kinase C. Experiments were performed based on single cell fluorescence imaging, fluorescence activated cell sorter/flow cytometer (FACS), scanning electron microscopy (SEM), atomic force microscopy (AFM) and dynamic light scattering (DLS). The released MVs were collected by differential centrifugation and characterized in both their size and zeta potential. Treatment of RBCs with 4-bromo-A23187 (positive control), lysophosphatidic acid (LPA), or phorbol-12 myristate-13 acetate (PMA) in the presence of 2 mM extracellular Ca2+ led to an alteration of cell volume and cell morphology. In stimulated RBCs, exposure of phosphatidylserine (PS) and formation of MVs were observed by using annexin V-FITC. The shedding of MVs was also observed in the case of PMA treatment in the absence of Ca2+, especially under the transmitted bright field illumination. By using SEM, AFM and DLS the morphology and size of stimulated RBCs, MVs were characterized. The sizes of the two populations of MVs were 205.8 ± 51.4 nm and 125.6 ± 31.4 nm, respectively. Adhesion of stimulated RBCs and MVs was observed. The zeta potential of MVs was determined in the range from - 40 mV to - 10 mV depended on the solutions and buffers used. An increase of intracellular Ca2+ or an activation of protein kinase C leads to the formation and release of MVs in human RBCs.

KW - red blood cells

KW - microvesicles

KW - phosphatidylserine

KW - fluorescence imaging

KW - cell adhesion

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Nguyen DB, Thuy Ly TB, Wesseling MC, Hittinger M, Torge A, Devitt A et al. Characterization of microvesicles released from human red blood cells. Cellular Physiology and Biochemistry. 2016 Mar 4;38(3):1085-1099. https://doi.org/10.1159/000443059