An improved 96 well plate format lipid quantification assay for standardization of experiments with extracellular vesicles

Abstract

The field of extracellular vesicles is an exponentially growing segment of biomedical sciences. However, the problems of normalisation and quantification of extracellular vesicle samples have not been completely solved yet. Currently, extracellular vesicle samples are standardized 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 extracellular vesicle concentration. Lipid bilayers are indispensable components of extracellular vesicles. Therefore, a lipidbased quantification, in combination with the determination of particle count and/or protein content, appears to be a straightforward and logical approach for the extracellular vesicle field. In this study we set the goal to improve the previously reported sulfo-phospho-vanillin total 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 optimized 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 sulfo-phospho-vanillin 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 extracellular vesicle standardisation. When using the optimised total lipid assay reported here, extracellular vesicle lipid measurements can be as easy as measuring proteins with a simple BCA test.