Characterization of EV subpopulation size and morphology.

<p>Apoptotic bodies (APO), microvesicles (MV) and exosomes (EXO) were isolated from conditioned tissue culture supernatant of BV-2 cells. Size distributions of the different subpopulations were assessed by tunable resistive pulse sensing (qNano) using three different membranes (NP200, NP400, and NP2000). Obtained particle concentrations were merged into single histograms for each EV subpopulation (left panels). Electron microscopic images of respective EV subpopulation pellets are shown (right panels).</p

Protein to lipid ratios of EV subpopulations.

<p>Protein to lipid ratios are presented for APOs, MVs, and EXOs isolated from MH-S (A), THP-1 (B), and BV-2 (C) cell lines (data represent ≥ 3 independent experiments for each EV type from all cell lines). Combined protein to lipid ratios obtained using EVs derived from MH-S, THP-1, BV-2, Jurkat, U937 as well as from human blood plasma are shown (D) (results of ≥ 12 independent experiments each EV type). Combined protein (E) and lipid concentrations (F) for the above cell line derived and blood plasma derived EVs are also shown. Mean values are represented by horizontal lines, and standard error means (SEMs) are indicated by error bars. The mean values ± SEM of lipid and protein concentrations (μg/mL) of conditioned media are reported below each respective EV subpopulation.</p

Flow cytometric characterization of EV subpopulations.

<p>EV subpopulations were isolated from BV-2 cells, and were analyzed along with the releasing cells either directly (MVs and APOs) or after coupling to latex beads (EXOs) by flow cytometry using annexin V FITC, as well as anti-CD9 FITC, anti-CD63 PE and anti-cholesterol CF488-conjugated antibodies, and Alexa Fluor647-conjugated cholera toxin (CTX) (all marked with thick black lines), and were compared to respective negative controls (thin black lines). Isotype controls were used for samples stained with fluorochrome-conjugated antibodies, whereas autofluorescence was detected in the absence of either annexin V or CTX. Images and figures are representatives of at least three independent experiments.</p

Spectral ratiometric determination of EV membrane lipid order.

<p>A shows the quantitative assessment of membrane lipid order in subpopulations of EVs secreted by BV-2 cells is shown (≥3 independent experiments each EV type). Ratiometric measurements of the fluorescent intensities at 560–600nm and at >660nm were carried out by confocal microscopy, and results are expressed as general polarization (GP) values. The higher GP value reflects a higher membrane lipid order. EXOs showed the highest order, while APOs and MVs were characterized by partially overlapping, intermediate order. B shows the flow cytometric determination of di-4-ANEPPDHQ staining of EV subpopulations secreted by BV-2 cells. Representative results of one out of n = 3 independent experiments. Left panels represent the fluorescence at 585±21 nm, while right panels represent fluorescence at 616±12 nm of unstained and stained vesicles and beads (thin and thick lines, respectively). Geometric mean fluorescent intensities (MFI) of unstained and stained APOs, MVs, EXOs coupled to latex beads, and beads without EXOs are shown (regular and bold text, respectively).</p

Protein to lipid ratio as a quality control parameter of EV preparations.

<p>A-C show results of 4 independent experiments where there were visible pellets for both the 20000g (MV) and 100,000g (“EXO”) preparations. While the normal protein to lipid ratio of MVs reflected a true vesicular pellet as also demonstrated by electron microscopy (B), a strongly elevated protein to lipid ratio suggested the absence of EXOs as was later confirmed by electron microscopy (C).</p