A protocol for loading Calcein-AM into extracellular vesicles from mammalian cells for clear visualization with a fluorescence microscope coupled to a deconvolution system.

Extracellular vesicles (EVs) are membrane-bound structures produced and released into the extracellular space by all types of cells. Due to their characteristics, EVs play crucial roles in cellular communication and signaling, holding an immense potential as biomarkers and molecular transporters. Va...

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Main Authors: María-Angélica Calderón-Peláez, Jaime E Castellanos, Myriam L Velandia-Romero
Format: Article
Language:English
Published: Public Library of Science (PLoS) 2025-01-01
Series:PLoS ONE
Online Access:https://doi.org/10.1371/journal.pone.0317689
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author María-Angélica Calderón-Peláez
Jaime E Castellanos
Myriam L Velandia-Romero
author_facet María-Angélica Calderón-Peláez
Jaime E Castellanos
Myriam L Velandia-Romero
author_sort María-Angélica Calderón-Peláez
collection DOAJ
description Extracellular vesicles (EVs) are membrane-bound structures produced and released into the extracellular space by all types of cells. Due to their characteristics, EVs play crucial roles in cellular communication and signaling, holding an immense potential as biomarkers and molecular transporters. Various methods have been developed to label and characterize EVs, however, visualizing EVs remains a process that requires highly specialized and expensive equipment, which is not always available in all the laboratories. In this study, we adapted a protocol originally designed for EVs analysis by flow cytometry using Calcein-AM, and convert it into a useful and effective tool for visualizing EVs by epifluorescence microscopy coupled with a deconvolution system. This approach can be very useful for basic EVs analyses, enabling researchers to verify their distribution and internalization across cells. Such insights can guide decisions on whether to advance to more detailed analysis using confocal microscopy or to perform additional assays.
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publishDate 2025-01-01
publisher Public Library of Science (PLoS)
record_format Article
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spelling doaj-art-c86222fd78ce40968256bc5a0178925b2025-02-01T05:30:50ZengPublic Library of Science (PLoS)PLoS ONE1932-62032025-01-01201e031768910.1371/journal.pone.0317689A protocol for loading Calcein-AM into extracellular vesicles from mammalian cells for clear visualization with a fluorescence microscope coupled to a deconvolution system.María-Angélica Calderón-PeláezJaime E CastellanosMyriam L Velandia-RomeroExtracellular vesicles (EVs) are membrane-bound structures produced and released into the extracellular space by all types of cells. Due to their characteristics, EVs play crucial roles in cellular communication and signaling, holding an immense potential as biomarkers and molecular transporters. Various methods have been developed to label and characterize EVs, however, visualizing EVs remains a process that requires highly specialized and expensive equipment, which is not always available in all the laboratories. In this study, we adapted a protocol originally designed for EVs analysis by flow cytometry using Calcein-AM, and convert it into a useful and effective tool for visualizing EVs by epifluorescence microscopy coupled with a deconvolution system. This approach can be very useful for basic EVs analyses, enabling researchers to verify their distribution and internalization across cells. Such insights can guide decisions on whether to advance to more detailed analysis using confocal microscopy or to perform additional assays.https://doi.org/10.1371/journal.pone.0317689
spellingShingle María-Angélica Calderón-Peláez
Jaime E Castellanos
Myriam L Velandia-Romero
A protocol for loading Calcein-AM into extracellular vesicles from mammalian cells for clear visualization with a fluorescence microscope coupled to a deconvolution system.
PLoS ONE
title A protocol for loading Calcein-AM into extracellular vesicles from mammalian cells for clear visualization with a fluorescence microscope coupled to a deconvolution system.
title_full A protocol for loading Calcein-AM into extracellular vesicles from mammalian cells for clear visualization with a fluorescence microscope coupled to a deconvolution system.
title_fullStr A protocol for loading Calcein-AM into extracellular vesicles from mammalian cells for clear visualization with a fluorescence microscope coupled to a deconvolution system.
title_full_unstemmed A protocol for loading Calcein-AM into extracellular vesicles from mammalian cells for clear visualization with a fluorescence microscope coupled to a deconvolution system.
title_short A protocol for loading Calcein-AM into extracellular vesicles from mammalian cells for clear visualization with a fluorescence microscope coupled to a deconvolution system.
title_sort protocol for loading calcein am into extracellular vesicles from mammalian cells for clear visualization with a fluorescence microscope coupled to a deconvolution system
url https://doi.org/10.1371/journal.pone.0317689
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