Dii dye

To trace LPS pre-Exo by fluorescent microscopy, they were labeled with DiI dye (Sigma) and washed in PBS with centrifugation at 100,000×g for 1 h at 4 °C. Then, the DiI-labeled LPS pre-Exo were co-cultured with THP-1 cells at a final concentration of 10 μg/ml. After 6 h, the cells were stained with Hoechst33342 for 8 min and washed with PBS. Finally, the cells were examined and photographed with a confocal imaging system (Olympus FV1200).

EVs were labelled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI) dye (Sigma-Aldrich), according to the manufacturer’s protocol. Briefly, 4 μL DiI dye was mixed with exosome suspension (100μg/ml) in diluent C and incubated for 10 min at 37°C. The labelling reaction was stopped by adding 20 ml chilled PBS. Labelled EVs were ultra-centrifuged at 100,000×g for 70 min, washed with PBS, ultra-centrifuged again at 100,000×g and the pellet was resuspended in PBS and stored at −80°C for further experiments. To assess the cell uptake of the EVs, N2a cells were seeded on coverslips in 12-well plates at a density of 1.5 × 10⁵ cells/well until 50% confluence was achieved and subsequently incubated with EVs labelled with DiI (0.71 ± 0.33 μg/μl from conditioned media) diluted in culture medium at 37°C for 6 h. In order to use equal amounts of EVs in the cell cultures, EVs protein content was quantitated by using BCA. Subsequently, cells were washed three times with PBS, fixed in 4% paraformaldehyde in PBS for 10 min, and mounted in DAPI mounting media. The cells were imaged using a Zeiss Axioplan-2 microscope (Carl Zeiss Microscope Systems, Toronto, ON, Canada) and AxioVision software (version 4.8) with identical photo settings.

Plasma RA-EVs and O₂-EVs obtained in Experiment 1 were labeled with non-lipophilic near IR dye (ExoGlow-Vivo EV Labeling Kit) as instructed by the manufacturer (Systems Biosciences)^{51 (link)}. The labeled EVs (50 µg/sample) or sham-labeled normal saline (NS) negative control were injected via the tail vein into normal neonatal rats on P7. Whole body imaging was done in vivo at 15 min, 1 h and 4 h after injection, and brain and lung tissues were dissected at 4 h for ex vivo imaging using an In Vivo Imaging system (PerkinElmer, Hopkinton, MA). In a separate experiment, RA-EVs and O₂-EVs were also labeled with DiI dye (Sigma, St. Louis, MO), and these EVs and sham-labeled NS were injected via the tail vein to a different sets of normal neonatal rats at P7. Brain tissues were collected 24 h later and tissue sections were examined by fluorescent microscopy for Dil signals. CSF was collected by tapping the cisternal magna and EVs were isolated from pooled CSF in each condition and analyzed by nanoparticle tracking.