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. 2022 Jan 24;18(4):1521–1538. doi: 10.7150/ijbs.66477

Figure 1.

Figure 1

Mass release of micrometer-sized extracellular vesicles in the early phase of ALI C57BL/6J mice induced by LPS. (A) Protocol to harvest micron-sized vesicles, microvesicles, and exosomes. BALF was harvested from mice instilled or not instilled with LPS, and extracellular vesicles were separated by serial centrifugation. The pellets were washed with PBS and then centrifuged twice to remove soluble factors or impurities outside the vesicles. (B) Representative transmission electron micrograph of extracellular vesicles pellets derived from differential centrifugation. Bar (from left to right) = 2 μm, 500 nm, 200 nm. (C) Protein concentration of three types of EVs and (D) the percentages of each type of EVs. (E) Immunostaining showing positive expression of FITC-Annexin V in the pellets. The pellets were incubated with FITC-labeled Annexin V+ for 15 min, washed twice with PBS, and observed under a fluorescence microscope. (F) The number of MsEVs was determined by flow cytometry. One- and 5-μm-diameter beads and 10-μm counting beads were used to gate 1-5 μm-sized EVs. Annexin V+ vesicles were counted as MsEVs (P2 * Annexin V+ rate / P1 * number of counting beads). Data are expressed as mean ± SD, n = 6. ***P < 0.001.