Figure 4.

Exosomes carry “garbage” to macrophages for disposal and disintegration. A) Schematic illustration of exosomes isolated from mouse plasma (with/without 2‐d fasting) labeled with DiD (hydrophobic fluorescent dye) for tracking. B) The same numbers of exosomes isolated from fasting or control groups, labeled with DiD dye (red) were injected into mice via the tail vein for in vivo imaging analysis. C) The mice were sacrificed and vein, liver, lung, heart, spleen, kidney, and brain tissues were collected and imaged. D) Confocal microscopic images of stained tissues. Liver, spleen, thymus, and lymph nodes were fixed with paraformaldehyde and frozen sections were prepared and stained with anti‐F4/80 (green), and nuclei were stained with DAPI (blue), with quantitative presentation of the fold‐changes of DiD‐positive foci in cells. Scale bars = 30 µm. A total of 60 randomly selected cells from three independent experiments were analyzed. E) Mice were injected with a macrophage scavenger and exosomes isolated from the plasma of mice fasted for 2 d (6‐months‐old) and labeled with DiD were injected into mice via the tail vein for in vivo imaging. F) The mice (n = 3) were sacrificed and liver, spleen, thymus, and lymph node tissues were collected and fixed with paraformaldehyde, and frozen sections were prepared and stained with anti‐F4/80 (green), and nuclei were stained with DAPI (blue), with quantitative presentation of the fold‐changes of DiD‐positive foci in cells. Scale bars = 30 µm. *p < 0.05. (G) Confocal microscopic images of stained cells. Primary macrophages were extracted from mouse bone marrow. Exosomes isolated from mouse plasma (with/without 2‐d fasting) labeled with DID (red) were cocultured with primary macrophages for 72 h, with quantitative presentation of fold‐changes of DiD‐positive foci in cells. Scale bars = 30 µm. A total of 60 randomly selected cells from three independent experiments were analyzed. H) Primary MEFs (passage 4) were cocultured with exosomes extracted from mice (with 2‐d fasting) and macrophages for 5 d. The proliferation ability of cells in each group was determined by CCK8 assay. Three independent experiments were analyzed. I) The cells were fixed and stained with anti‐γH2AX (green) and DAPI staining for nuclei (blue), with quantitative presentation of the fold‐change of γH2AX‐positive foci in cells. A total of 60 randomly selected cells from three independent experiments were analyzed. Scale bars = 30 µm. *p < 0.05.