Fm4 64

Protein concentration in the MV suspensions was determined using a Bradford assay, with bovine serum albumin as the standard. Lipid concentration in the MV suspensions was determined using FM4‐64 dye (Life Technologies, Carlsbad, CA, USA), with water‐soluble linoleic acid (Sigma, St. Louis, MO, USA) as the standard. Standards and samples (5 µl) were mixed with 100 µl of 5 µg ml⁻¹ FM4‐64 in the 96‐well black plate, and incubated for 10 min at room temperature. Fluorescence from FM4‐64 (excitation at 535 nm and emission at 625 nm) was detected with the plate reader Cytation5 (Biotek, Winooski, VT, USA). Limulus assay was performed to quantify LPS using an Endospecy ES‐50M kit (Seikagaku Co., Tokyo, Japan) according to the manufacturers’ instructions, with LPS from Escherichia coli O111:B4 (Sigma) as the standard. MV particle size was measured from TEM images using the Fiji image processing package (a variant of ImageJ) (Schindelin et al., 2012).

The surface of a 384-well black plate (Greiner, Kremsmünster, Austria) was incubated with 20 μL of 0.01% poly-l-lysine (Sigma-Aldrich) at 20 °C for 1 h, followed by washing twice with 50 μL of DPBS (Thermo Fisher Scientific). Twenty microliters of purified EVs or EV-free supernatants were applied to the wells and incubated at 37 °C for 24 h, followed by lipid-staining with 20 μL of 5 μg mL⁻¹N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM4-64, Thermo Fisher Scientific) at 20 °C for 30 min in the dark. The fluorescence intensity of FM4-64 was measured at 515/20 nm excitation and 635/20 nm emission wavelength using a Cytation 5 cell imaging multi-mode reader (BioTek, Winooski, VT, United States). A non-coated plate was used with EVs as a control.