Accumulation of exosomes secreted from macrophages in Cath.A neurons and genetic material transfer.

<p><b>A</b>: Cath.A neurons grown on slides were fixed and stained with Anti-NeuN Antibodies (blue, left picture); exosomes were isolated from Raw 264.7 macrophages media, stained with lipophilic fluorescent dye, DIO (green), and added to Cath.A neurons for 24 hours (right picture). <b>B</b>: Raw 264.7 macrophages were transfected with fluorescently-labeled with YOYO-1 tomato protein <i>p</i>DNA (green), and then cultured in complete media. Confocal images of transfected macrophages on day 3 show incorporation of <i>p</i>DNA (green) in the nucleus and expression of tomato protein (red) in the cytoplasm. <b>C</b>: Media from macrophages transfected as described above with tomato protein <i>p</i>DNA (labeled with YOYO-1) was collected over 24 hours, and isolated exosomes were added to Cath.A neurons for various times. Then, the neurons were fixed and stained with Anti-NeuN Antibodies (blue). Confocal images of neurons incubated with exosomal fraction demonstrated relatively constant amount of YOYO-1-labeled <i>p</i>DNA (green), and increasing in time expression levels of tomato protein (red) confirmed by the quantification of green and red fluorescence on confocal images (graph). Co-localization of YOYO-1-labeled genetic material and expressed tomato protein in neurons is manifested by yellow staining. Statistical significance of tomato protein expression levels (shown by asterisk: <i>p</i><0.05) was assessed by a standard t-test compared to day one after transfection. The bar: 20 µm.