An improved 96 well plate format lipid quantification assay for standardisation of experiments with extracellular vesicles
Authors: Tam√°s Visnovitz
Xabier Osteikoetxea
Krisztina V Vukman
Eszter √Ågnes T√≥th
Robert Horv√°th
Edit I Buz√°s
Inna Sz√©k√°cs
Barbara W S√≥dar
Zolt√°n Varga
Judith Mih√°ly
Anna Koncz
P√©ter L≈ërincz
Journal: Journal of Extracellular Vesicles (2019)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: EV; apoptotic bodies; exosomes; large oncosomes; lipid assay
Affiliations: Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary, Institute of Materials and Environmental Chemistry, Research Centre for Natural Sciences of the Hungarian Academy of Sciences, Budapest, Hungary, Department of Anatomy, Cell and Developmental Biology, E√∂tv√∂s Lor√°nd University, Budapest, Hungary, Nanobiosensorics Laboratory MTA-EK-MFA, Budapest, Hungary, MTA-SE Immune-Proteogenomics Extracellular Vesicle Research Group, Budapest, Hungary

Abstract:  The field of extracellular vesicles (EVs) is an exponentially growing segment of biomedical sciences. However, the problems of normalisation and quantification of EV samples have not been completely solved. Currently, EV samples are standardised on the basis of their protein content sometimes combined with determination of the particle number. However, even this combined approach may result in inaccuracy and overestimation of the EV concentration. Lipid bilayers are indispensable components of EVs. Therefore, a lipid-based quantification, in combination with the determination of particle count and/or protein content, appears to be a straightforward and logical approach for the EV field. In this study, we set the goal to improve the previously reported sulfo-phospho-vanillin (SPV) lipid assay. We introduced an aqueous phase liposome standard (DOPC) to replace the purified lipid standards in organic solvents (used commonly in previous studies). Furthermore, we optimised the concentration of the vanillin reagent in the assay. We found that elimination of organic solvents from the reaction mixture could abolish the background colour that interfered with the assay. Comparison of the optimised assay with a commercial lipid kit (based on the original SPV lipid assay) showed an increase of sensitivity by approximately one order of magnitude. Thus, here we report a quick, reliable and sensitive test that may fill an existing gap in EV standardisation. When using the optimised lipid assay reported here, EV lipid measurements can be more reliable than protein-based measurements. Furthermore, this novel assay is almost as sensitive and as easy as measuring proteins with a simple BCA test. 

Retracted: No

PubPeer comments:
 Possible repeated image in Figure 4A. ***  , 
 Dear Actinopolyspora Biskrensis, Thank you very much for bringing the issue to our attention. We solemnly apologize for using the wrong picture in Fig4a. When typesetting and adding auxiliary lines, we made the mistake of using the image of lira+Met (0 h) twice without showing the correct image of Met (0 h). We have found this error in June 2019, and made corrigendum to it. We resent the corrected figure to PLoS One in July 2019, and provided all the relevant original data to the editor at that time. The corrected figure is shown as below. All the relevant images of Fig4a (n=4) are shown as below. , 
 #2 Thank you for replying here. I do not see that a correction has been published. Do you have any correspondence from the journal that explains why they apparently did not respond for over a year and a half? , 
 Dear Actinopolyspora Biskrensis, Thank you very much. We contacted the journal editor twice in 2019, but there was no further response about the process of publishing our corrigendum. Weu00a0willu00a0further contact the Journal aboutu00a0theu00a0issue. ,