Method for determining the lipid content of extracellular vesicles

Method for quantification of lipids in a sample comprising extracellular vesicles (EVs), the method comprising the step of

• a) providing an EV preparation from the sample, the EV preparation being an aqueous suspension comprising EVs;
• b) contacting the EV preparation of step a) with sulphuric acid and subsequently with phospho-vanillin reagent and then allowing a colour reaction to develop, to obtain a coloured solution;
• c) measuring the absorbance of the coloured solution of step b);
• d) comparing the absorbance measured in step c) to the absorbance of one or more reference suspension and calculating the amount of lipids in the EV preparation from the result of said comparison;
• characterized in that
• the EV sample preparation in step a) is substantially free of organic solvents;
• the reference suspension in step d) comprises liposomes suspended in aqueous solvent, wherein the liposomes are formed from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and wherein the aqueous solvent is an aqueous buffer selected from PBS, NaCl-HEPES, MES, TRIS, physiological NaCl solution or DPBS;
• and
• the reference suspension is substantially free of organic solvents.

Method for quantification of biological membranes in a sample comprising

• a) providing a preparation from the sample, the preparation comprising biological membranes in an aqueous suspension;
• b) contacting the preparation of step a) with sulphuric acid and subsequently with phospho-vanillin reagent and then allowing a colour reaction to develop, to obtain a coloured solution;
• c) measuring the absorbance of the coloured solution of step b) to obtain a preparation absorbance value, and measuring the absorbance of one or more reference solution prepared from a reference suspension to obtain a reference absorbance value;
• d) comparing the preparation absorbance value and the one or more reference absorbance value measured in step c) to the absorbance of one or more reference suspension and calculating the amount of lipids in the preparation from the result of said comparison;
• e) calculating the amount of membranes in the preparation of step a) from the amount of lipids calculated in step d);
• characterized in that
• the EV sample preparation in step a) is substantially free of organic solvents;
• the reference suspension in step d) comprises liposomes suspended in aqueous solvent, wherein the liposomes are formed from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and wherein the aqueous solvent is an aqueous buffer selected from PBS, NaCl-HEPES, MES, TRIS, physiological NaCl solution or DPBS;
• and
• the reference suspension is substantially free of organic solvents.