Abstract
Extracellular vesicles (EVs) are constantly secreted from both eukaryotic and prokaryotic cells. EVs, including those referred to as exosomes, may have an impact on cell signaling and an incidence in diseased cells. In this manuscript, a platform to capture, quantify, and phenotypically classify the EVs secreted from single cells is introduced. Microfluidic chambers of about 300 pL are employed to trap and isolate individual cells. The EVs secreted within these chambers are then captured by surface-immobilized monoclonal antibodies (mAbs), irrespective of their intracellular origin. Immunostaining against both plasma membrane and cytosolic proteins was combined with highly sensitive, multicolor total internal reflection fluorescence microscopy to characterize the immobilized vesicles. The data analysis of high-resolution images allowed the assignment of each detected EV to one of 15 unique populations and demonstrated the presence of highly heterogeneous phenotypes even at the single-cell level. The analysis also revealed that each mAb isolates phenotypically different EVs and that more vesicles were effectively immobilized when CD63 was targeted instead of CD81. Finally, we demonstrate how a heterogeneous suppression in the secreted vesicles is obtained when the enzyme neutral sphingomyelinase is inhibited.
Highlights
Extracellular vesicles (EVs) are constantly secreted from both eukaryotic and prokaryotic cells
Our focus is on collecting EVs secreted by single cells and performing their phenotype-specific classification (Fig. 1A)
The enrichment and classification of EVs usually rely on their physical properties, like density and diameter
Summary
Extracellular vesicles (EVs) are constantly secreted from both eukaryotic and prokaryotic cells. The EVs secreted within these chambers are captured by surface-immobilized monoclonal antibodies (mAbs), irrespective of their intracellular origin Immunostaining against both plasma membrane and cytosolic proteins was combined with highly sensitive, multicolor total internal reflection fluorescence microscopy to characterize the immobilized vesicles. The regulatory effect of exosomes, owed to the bioactive nature of their content (e.g., membrane proteins [Fig. 1A], nucleic acids, carbohydrates, and lipids), has boosted the identification of their biological purpose and function on physiological and pathological conditions, such as diabetes, cancer, and neurodegenerative diseases [6,7,8,9] These vesicles have attracted interest as putative biomedical diagnostic tools or therapeutic drug vehicles [10,11,12,13]. This will give further insights into the most important cues of cell–cell interactions besides direct contact with other cells and the extracellular matrix and, most of all, signaling factors, which are commonly in the focus of single-cell studies
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