Confocal microscopy images of fibroblasts incubated with microvesicles/exosomes stained with acridine orange.

<p>Confocal microscopy picture of DNA-stained microvesicles/exosomes after dialysis, ultracentrifugation and resuspension in DMEM. After incubation with fibroblasts for 3 h at 37°C the DNA-staining localizes in fibroblasts to and inside the nuclear membrane. Additional light microscopy was used to add a layer in images to visualize cell borders. Arrows in A) and B) indicate acridine orange staining inside nuclei. B) also visualizes red wave length which detects acridine orange staining for RNA. Yellow staining shows colocalization of DNA and RNA.</p

Transmission electron microscopy of purified microvesicles/exosomes.

<p>A) Microvesicles/exosomes displaying an electron dense appearance, and B) electron lucent appearance. Bar represents 100 nm.</p

Filtering of differentially expressed genes.

<p>Fibroblasts incubated for 48 h with Claycomb medium, previously incubated for 24 h with cardiomyocytes were compared to fibroblasts incubated with fresh Claycomb medium.</p><p>Fibroblasts incubated for 48 h with supernatant from ultracentrifuged Claycomb medium, previously incubated for 24 h with cardiomyocytes were compared to fibroblasts incubated with fresh Claycomb medium.</p><p>Fibroblasts incubated for 48 h with pellet from ultracentrifuged Claycomb medium, previously incubated for 48 h with cardiomyocytes. Pellet was dissolved in DMEM and compared to fibroblasts incubated with fresh DMEM.</p><p>False Discovery Rate (FDR) was used for corrections for multiple testing. Significant up-regulation was defined as a foldchange >1.5 and significant down-regulation was defined as foldchange <0.67. A minimum signal intensity value of 50 was utilized. Abbreviations: Cm, cardiomyocytes; Fb, fibroblasts; sup. supernatant after ultracentrifugation; Avg. sign., average signal; FDR, False Discovery Rate; ↑, up-regulated; ↓, down-regulated; DMEM, Dulbecco's modified Eagle's medium.</p

Flow cytometry of DNA-stained microvesicles/exosomes.

<p>A) Enhanced fluorescence at the 530±15 nm channel of membrane permeable acridine orange-stained microvesicles/exosomes (below) in comparison with unstained microvesicles/exosomes (above). B) Weak or no fluorescence at 670 nm/LP channel of membrane impermeable propidium iodide-stained microvesicles/exosomes (below) not differing from unstained microvesicles/exosomes (above).</p

Detection of proteins on microvesicle/exosome surface with flow cytometry.

<p>Microvesicles/exosomes prepared from Claycomb culture medium was incubated with antibodies conjugated with phycoerythrin (PE). A) Mouse anti-caveolin-3, was detected on approximately 30% of the microvesicles/exosomes. B) Mouse anti-flotillin-1, was detected on approximately 80% of the microvesicles/exosomes. C) Mouse anti-annexin-2, was not detected on the microvesicles/exosomes. D) Mouse anti-clathrin heavy chain, was not detected on the microvesicles/exosomes. The distribution of exosomes presenting caveolin-3 and flotillin-1 indicates that the sample contains more than one population of microvesicles/exosomes.</p