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

Grupos de investigación

• Grupo de Virología

  

  Investigador

  Jaime Eduardo Castellanos Parra

Resumen

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. © 2025 Calderón-Peláez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Keywords

• Animals; Extracellular Vesicles; Flow Cytometry; Fluoresceins; Fluorescent Dyes; Humans; Microscopy, Fluorescence; calcein; calcein AM; fluorescein derivative; fluorescent dye; adult; animal cell; animal experiment; animal husbandry; animal model; animal welfare; Article; Bagg albino mouse; behavior; body weight; cervical spine dislocation; confocal microscopy; controlled study; deconvolution; distress syndrome; epifluorescence microscopy; exosome; extracellular space; female; flow cytometry; fluorescence microscopy; image analysis; male; mammal; mammal cell; mouse; nanoparticle tracking analysis; nonhuman; animal; chemistry; human; metabolism; procedures