Evaluation of neutral lipid composition of EVs. (A) High performance thin liquid chromatography for neutral lipids of the EVs from Acanthamoeba castellanii. Lane 1 – Lipid standards (TG- Triglycerides −; DAG- Diacylglycerol; MAG- Monoacylglycerol), Lane 2- EVs secreted in PYG, Lane 3- EVs secreted in glucose medium and Lane 4- Lipid standards (EC- Esterified cholesterol; FFA- Free fatty acids; S- cholesterol; PL/O- Phospholipids. (B) GC-MS for determination of sterol composition in EVs of A. castellanii. Two biological replicates were analyzed with similar results. Peaks of interest are represented by numerals, followed by their retention time in the chromatogram. Peak 1 – [(3-β)-cholest-5-en-3-yl]oxy]trimethyl]-silane (rt = 32.557 min), Peak 2 – [(ergosta-5,7,22-trien-3β-yloxi)trimethyl]-silane (rt = 35.355 min) and Peak 3 -stigmasta-5,7,22-trien-3α-ol (rt = 35.520 min). (C) GC-MS for fatty acids present in the EVs of A. castellanii. Peaks of interest are represented by numerals, followed by their retention time in the chromatograph. Peak 1- methyl miristate (rt = 20.663 min), Peak 2- methyl palmitate (rt = 32.008 min), Peak 3- methyl linoleate (rt = 44.200 min), Peak 4- methyl oleate (rt = 44.683 min), Peak 5- methyl stearate (rt = 46.675 min) of and Peak 6- methyl erucate (rt = 75.033 min).