2.2. Quantifying EV concentration
The EV yield was quantified by two different measures as is standard
according to the MISEV2018 guidelines. Firstlly, the total protein
concentration was measured using the BCA protein assay kit (Thermo
Fisher Scientific). Next, total lipid concentration was quantified using
a lipid quantification kit (STA-613, Cell Biolabs, San Diego, CA)
previously used by other groups investigating EVs [36]. Briefly, 15
µL of each EV group were added to a 96-well plate and incubated
uncovered for 30 min at 90°C. The samples were then transferred to 4°C
for 5 min before adding 150 µL of 18M sulfuric acid. They were then
incubated at 90°C for 10 min and again transferred to 4°C for 5 min.
Next, 100 µL of each sample was transferred to a clean 96-well plate
before being read at 540 nm to determine the background. 100 µL of
Vanillin Reagent was then carefully mixed and incubated at 37°C for 15
min before reading at 540 nm to determine the signal. The background was
subtracted from the signal and compared to a previously prepared
standard curve.