FIGURE 3.

Di8 ANEPPS detects the majority of Triton‐X‐ solublizable EVs in fraction 3 from human milk. Fractions 2 (A, C) and 3 (B, D) were isolated from human milk sample (c), concentrated using Amicon^® concentration column (10 kDa MWCO), and labeled with Di8‐ANEPPS. Aliquots of the labeled sample were then incubated with either 0.1% Triton‐X100 (final concentration), 5 mM EDTA (final concentration), 50 µg/mL proteinase K (Thermo Fisher Scientific, final concentration), or 0.1% Triton‐X100 + 50 µg/mL proteinase K before particle counts were measured by nanoparticle tracking analysis (NTA). (a & b) show the Di8 particle measurements for comparison with scatter, while (c & d) show measurements detected with the fluorescence reading alone. Data represent the average of triplicate runs for fractions 2 and 3 of a single milk sample. Data are smoothed using GraphPad Prism® v10.