Exoglow vivo ev labeling kit

MSC EVs or PL labeled with a fluorescent dye (ExoGlow -Vivo EV Labeling Kit, System Biosciences, Palo Alto, CA) were injected with IT on P3. Saline incubated with free dye and processed identically as labeled EVs was used as PL. The animals were imaged at various time points following IT injection of labeled EVs and PL using an in vivo imaging system (IVIS in-vivo Imaging System; PerkinElmer, Waltham, MA). Rats injected with labeled EVs or PL were also euthanized 90 min after injection and organs were removed and imaged under IVIS.

The lipophilic dye DiI (2 μM) was gently mixed with GC-VLNs in PBS for 30 min at 37 ºC to label the membrane lipids. 35-fold more PBS was added to the mixture, and it was subjected to ultracentrifugation at 100,000 ×g for 2 h at 4 ºC to remove free dye. The obtained GC-VLNs were resuspended in PBS and incubated at 37 ºC for 1 h, ready to be incubated with BMDMs. Proteins and RNAs inside GC-VLNs were labeled with ExoGlow protein EV labeling kit and ExoGlow RNA EV labeling kit (System Biosciences), respectively, per manufacturer's protocols. BMDMs were incubated with the fluorescence-labeled GC-VLNs for 16 h or the indicated time in the time-course experiment, washed with PBS 4 times, and fixed with 4% paraformaldehyde (Sigma). Images were taken using an A1R-Ti2 confocal system (Nikon, Melville, NY, USA).
For the distribution studies of GC-VLNs in mice, fluorescence dye from ExoGlow-Vivo EV labeling kit (EXOGV900A-1, System Biosciences) was used to label GC-VLNs at the ratio of 60×10¹⁰ nanoparticles : 1 μl dye, per manufacturer's protocol. The labeled GC-VLNs were resuspended in 30 mL of PBS and ultra-centrifuged at 100,000 ×g for 2 h at 4 ºC to remove the free dye. The wash step was repeated two times. The obtained GC-VLNs covalently linked to the dye were given to mice through oral gavage or intravenous injection.

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.

Pregnant Sprague-Dawley rats were purchased from Charles River Laboratory (Wilmington, MA). The following antibodies were used for immunostaining, immunofluorescence staining, Western blot analysis and FACS analysis: rabbit anti-GSDMD and mouse anti-AIF-1 from Novusbio (Littleton, CO); rabbit anti-pro-SPC and mouse anti-CD63 from EDMillipore (Temecula, CA); mouse anti-CD9 antibody from ThermoFisher (Waltham, MA); rabbit anti-Ki67 from Abcam (Cambridge, MA); mouse anti-vonWillebrand factor (vWF) from Dako (Carpinteria, CA); rabbit anti-SPC-PE from Biossusa (Woburn, MA). Total Exosome Isolation Kit was obtained from ThermoFisher. Exo-FLOW Exosome Purification Kit and ExoGlow-Vivo EV Labeling Kit were obtained from Systems Biosciences (Palo Alto, CA). The lipophilic fluorescent dye, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarocyanine perchlorate (DiI) was purchased from Sigma (Louis, MO). MLE-12 cells were obtained from ATCC (Manassas, VA), pulmonary vascular endothelial cells (PVEC) were obtained from Lonza (Walkersville, MD), and rat fetal neural stem cells (NSC) were obtained from ThermoFisher. The primers for all the qRT-PCR were obtained from ThermoFisher.

Mice were fed an alfalfa-free diet to prevent auto fluorescence of tissues (Perkin-Elmer personal communication). Exosomes were labeled with ExoGlow-Vivo EV Labeling Kit (System Biosciences, Palo Alto, CA). Labeled exosomes or PBS containing exosomes were injected via tail vein into 17- to 18-month-old C57BL/6 mice. Mice were anesthetized with isoflurane and imaged using an IVIS Spectrum In Vivo Imaging System (Perkin Elmer). The mice were imaged at 5, 10, 15, 30, 60, 90 min, 2, 4, 6, and 24 hr as well as at 2, 3, and 19 days post injection. Imaging data was analyzed using Living Image Software (Perkin Elmer). Control experiments were conducted with dye alone.