Exosparkler exosome membrane labeling kit green

Labeling of exosomes was performed with ExoSparkler Exosome Membrane Labeling Kit-Green (Dojindo, Kumamoto, Japan) following the manufacturer’s instructions. Following intranasal administration of exosomes, animals were sacrificed at 1, 3, and 24 h post-administration. Rats were deeply anesthetized and transcardially perfused with PBS followed by 4% paraformaldehyde (PFA), as previously reported [18 (link)]. The brains were then extracted, and fixed in 4% PFA for 24 h, embedded in paraffin, and sectioned into coronal slices of 4 μm in thickness. Serial sections of the olfactory nerve (15 mm anterior to the bregma), olfactory tract (5 mm anterior to the bregma), striatum (0 mm anterior to the bregma), and midbrain (7 mm posterior to the bregma) were prepared for the biodistribution analysis of labeled exosomes, with four non-overlapping regions of interest (ROI) set for each section. Positive fluorescence signals indicative of labeled exosomes were quantified using an automated cell/area counter according to the manufacturer’s instruction (BZ-X Analyzer, Keyence Co., Osaka, Japan). To eliminate autofluorescence signals in each area, the average fluorescence observed in the control group for each area was subtracted from the specimen.

Commercially available exosomes, bovine milk exosome (mean diameter: 119 nm, EXBM100L, COSMO Bio, Co., Ltd., Tokyo, Japan), human breast milk exosome (mean diameter: 174 nm, EXHM100L, COSMO Bio, Co., Ltd., Tokyo, Japan), and human breast cancer (MCF-7) exosome (peak diameter: 100 nm, EXOP-100A-1, System Biosciences, LLC, Palo Alto, CA, USA), were used. The exosomes were fluorescently labeled using ExoSparkler Exosome Membrane Labeling Kit-Green or Deep Red (Dojindo Laboratories, Kumamoto, Japan). Before the DEP experiments, the exosome suspensions were desalted using Amicon Ultra 0.5, 10 k (Merck KGaA, Darmstadt, Germany). The conductivity of the suspension medium was adjusted using NaCl solution.