Analyzing Extracellular Vesicle‐associated DNA Using Transmission Electron Microscopy at the Single EV‐level DOI Creative Commons
Thupten Tsering, Amélie Nadeau, Janusz Rak

и другие.

Current Protocols, Год журнала: 2024, Номер 4(11)

Опубликована: Ноя. 1, 2024

Abstract Extracellular vesicles (EVs) play an important role in cell‐cell communication, carrying bioactive molecules including DNA. EV‐associated DNA (EV‐DNA) has created enormous interest the field of biomarkers, particularly related to liquid biopsy. However, its analysis is challenging due nanoscale structure EVs, low abundance EV‐DNA, and surrounding biogenetic debate. Therefore, novel protocols enhance accurate detection EV‐DNA are essential study normal physiology disease states. Here, we provide two for confirming presence from biological samples. In first protocol, ultrathin sectioning EVs combined with immunogold labeling detect double‐stranded (ds) within EV lumen using transmission electron microscopy (TEM). second whole‐mount allows detailed morphological their surface‐associated Using TEM imaging, have demonstrated that cancer‐cell‐derived individual exhibit simultaneous positivity dsDNA surface protein tetraspanin 9. We believe this method can be used label any proteins inside as well on EVs. This aid characterization single identification verification biomarkers. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 : isolation cell‐culture‐conditioned medium, embedding, sectioning, labeling, imaging 2 Whole‐mount immunolabeling

Язык: Английский

Analyzing Extracellular Vesicle‐associated DNA Using Transmission Electron Microscopy at the Single EV‐level DOI Creative Commons
Thupten Tsering, Amélie Nadeau, Janusz Rak

и другие.

Current Protocols, Год журнала: 2024, Номер 4(11)

Опубликована: Ноя. 1, 2024

Abstract Extracellular vesicles (EVs) play an important role in cell‐cell communication, carrying bioactive molecules including DNA. EV‐associated DNA (EV‐DNA) has created enormous interest the field of biomarkers, particularly related to liquid biopsy. However, its analysis is challenging due nanoscale structure EVs, low abundance EV‐DNA, and surrounding biogenetic debate. Therefore, novel protocols enhance accurate detection EV‐DNA are essential study normal physiology disease states. Here, we provide two for confirming presence from biological samples. In first protocol, ultrathin sectioning EVs combined with immunogold labeling detect double‐stranded (ds) within EV lumen using transmission electron microscopy (TEM). second whole‐mount allows detailed morphological their surface‐associated Using TEM imaging, have demonstrated that cancer‐cell‐derived individual exhibit simultaneous positivity dsDNA surface protein tetraspanin 9. We believe this method can be used label any proteins inside as well on EVs. This aid characterization single identification verification biomarkers. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 : isolation cell‐culture‐conditioned medium, embedding, sectioning, labeling, imaging 2 Whole‐mount immunolabeling

Язык: Английский

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