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

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.

En bloc release of MVB‐like small extracellular vesicle clusters by colorectal carcinoma cells

“…Contrast and brightness of electron micrographs were edited by Adobe Photoshop CS3 (Adobe Photoshop Incorporation, CA). For immune EM of released EVs, intact EVs were enriched by 2,000 g centrifugation. During sample preparation, we have followed the basic protocol described previously by Visnovitz et al [12] with minor modifications. Two to three µL of EV sample in 0.22 µm filtered PBS was applied onto the surface of a 300 mesh formvar‐coated Ni grid and was incubated for 10 min at room temperature.…”

Extracellular vesicles from human iPSC‐derived neural stem cells: miRNA and protein signatures, and anti‐inflammatory and neurogenic properties

“…We measured the amount of total protein in aliquots of EVs containing 5, 10, 20 and 50 × 10 9 EVs using the BCA protein assay kit (Thermo Fisher Scientific) and determined EV numbers per microgram of protein. The total lipid content in EV preparations was measured by the modified sulpho-phospho vanillin (SPV) method, as described elsewhere [27]. Briefly, both standard (1,2-Dioleoyl-sn-glycero -3-phosphocholine, DOPC; Sigma, St. Louis, MO, USA) and hiPSC-NSC-EV samples in SEC buffer (40 μl each) were sonicated at 35 amplitude (20 kHz) for 10 minutes, intensely vortexed for 2 minutes and mixed with 200 μl of 96% sulphuric acid.…”

Framework for Rapid Comparison of Extracellular Vesicle Isolation Methods

“…Samples from each isolation method are then loaded into a 96-well plate into a Quanterix HD-X instrument for Simoa analysis of CD9, CD63, CD81, and albumin. (7)(8)(9)(10) and then averaging the three tetraspanin percentages. e. Albumin levels using different EV isolation methods from plasma.…”

“…Since biofluids, and plasma in particular, contain an abundance of lipoproteins and protein aggregates at levels higher than those of EVs (6,7), these methods are illsuited for quantifying EVs (2). Lipid dyes have also been used to label and measure EVs (8,9), but these dyes also bind to lipoproteins, however, and lack sensitivity (2).…”