TY - JOUR
T1 - A cell-based assay for CD63-containing extracellular vesicles
AU - Cashikar, Anil G.
AU - Hanson, Phyllis I.
N1 - Funding Information:
This work was supported by R01 GM076686 and GM122434 from the National Institutes of Health to P.I.H. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Teri Naismith for general lab maintenance; Dr. Joshua Rubin and Dr. Gerry Linette for help with cell lines; Dr. Philip Stahl for helpful discussions.
Publisher Copyright:
© 2019 Cashikar, Hanson. 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.
PY - 2019/7/1
Y1 - 2019/7/1
N2 - Extracellular vesicles (EVs) are thought to be important in cell-cell communication and have elicited extraordinary interest as potential biomarkers of disease. However, quantitative methods to enable elucidation of mechanisms underlying release are few. Here, we describe a cell-based assay for monitoring EV release using the EV-enriched tetraspanin CD63 fused to the small, ATP-independent reporter enzyme, Nanoluciferase. Release of CD63-containing EVs from stably expressing cell lines was monitored by comparing luciferase activity in culture media to that remaining in cells. HEK293, U2OS, U87 and SKMel28 cells released 0.3%-0.6% of total cellular CD63 in the form of EVs over 5 hrs, varying by cell line. To identify cellular machinery important for secretion of CD63-containing EVs, we performed a screen of biologically active chemicals in HEK293 cells. While a majority of compounds did not significantly affect EV release, treating cells with the plecomacrolides bafilomycin or concanamycin, known to inhibit the V-ATPase, dramatically increased EV release. Interestingly, alkalization of the endosomal lumen using weak bases had no effect, suggesting a pH-independent enhancement of EV release by V-ATPase inhibitors. The ability to quantify EVs in small samples will enable future detailed studies of release kinetics as well as further chemical and genetic screening to define pathways involved in EV secretion.
AB - Extracellular vesicles (EVs) are thought to be important in cell-cell communication and have elicited extraordinary interest as potential biomarkers of disease. However, quantitative methods to enable elucidation of mechanisms underlying release are few. Here, we describe a cell-based assay for monitoring EV release using the EV-enriched tetraspanin CD63 fused to the small, ATP-independent reporter enzyme, Nanoluciferase. Release of CD63-containing EVs from stably expressing cell lines was monitored by comparing luciferase activity in culture media to that remaining in cells. HEK293, U2OS, U87 and SKMel28 cells released 0.3%-0.6% of total cellular CD63 in the form of EVs over 5 hrs, varying by cell line. To identify cellular machinery important for secretion of CD63-containing EVs, we performed a screen of biologically active chemicals in HEK293 cells. While a majority of compounds did not significantly affect EV release, treating cells with the plecomacrolides bafilomycin or concanamycin, known to inhibit the V-ATPase, dramatically increased EV release. Interestingly, alkalization of the endosomal lumen using weak bases had no effect, suggesting a pH-independent enhancement of EV release by V-ATPase inhibitors. The ability to quantify EVs in small samples will enable future detailed studies of release kinetics as well as further chemical and genetic screening to define pathways involved in EV secretion.
UR - http://www.scopus.com/inward/record.url?scp=85069826056&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0220007
DO - 10.1371/journal.pone.0220007
M3 - Article
C2 - 31339911
AN - SCOPUS:85069826056
SN - 1932-6203
VL - 14
JO - PLoS ONE
JF - PLoS ONE
IS - 7
M1 - e0220007
ER -