Journal Article


Confocal microscopy analysis reveals that only a small proportion of extracellular vesicles are successfully labelled with commonly utilised staining methods

Abstract

Assessing genuine extracellular vesicle (EV) uptake is crucial for understanding the functional roles of EVs. This study measured the bona fide labelling of EVs utilising two commonly used fluorescent dyes, PKH26 and C5-maleimide-Alexa633. MCF7 EVs tagged with mEmerald-CD81 were isolated from conditioned media by size exclusion chromatography (SEC) and characterised using Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM), MACsPlex immunocapture assay and immunoblots. These fluorescently tagged EVs were subsequently stained with C5-maleimide-Alexa633 or PKH26, according to published protocols. Colocalisation of dual-labelled EVs was assessed by confocal microscopy and quantified using the Rank-Weighted Colocalisation (RWC) algorithm. We observed strikingly poor colocalisation between mEmerald-CD81-tagged EVs and C5-Maleimide-Alexa633 (5.4% ± 1.8) or PKH26 (4.6% ± 1.6), that remained low even when serum was removed from preparations. Our data confirms previous work showing that some dyes form contaminating aggregates. Furthermore, uptake studies showed that maleimide and mEmerald-CD81-tagged EVs can be often located into non-overlapping subcellular locations. By using common methods to isolate and stain EVs we observed that most EVs remained unstained and most dye signal does not appear to be EV associated. Our work shows that there is an urgent need for optimisation and standardisation in how EV researchers use these tools to assess genuine EV signals.

Attached files

Authors

Melling, Genevieve E.
Conlon, Ross
Pantazi, Paschalia
Dellar, Elizabeth R.
Samuel, Priya
Baena-Lopez, Luis Alberto
Simpson, Jeremy C.
Carter, David R.F.

Oxford Brookes departments

Department of Biological and Medical Sciences

Dates

Year of publication: 2022
Date of RADAR deposit: 2022-03-30


Creative Commons License This work is licensed under a Creative Commons Attribution 4.0 International License


Related resources

This RADAR resource is Identical to Confocal microscopy analysis reveals that only a small proportion of extracellular vesicles are successfully labelled with commonly utilised staining methods

Details

  • Owner: Joseph Ripp
  • Collection: Outputs
  • Version: 1 (show all)
  • Status: Live