Exoglow rna ev labeling kit

Bovine milk sEVs (BEVs) were isolated from skim milk from a local grocery store by using sequential ultracentrifugation and authenticated by using Nanosight NS300 nanoparticle size analysis, scanning electron microscopy and transmission electron microscopy as previously described (Supplementary Figure 1) (32 (link)). The antibodies and their dilutions used in immunoblot analysis were the same as previously described (32 (link)). Protocol details were deposited in the EV-Track database (ID EV210338). BEVs were suspended in sterile phosphate-buffered saline (PBS) and kept at −80°C until use. For transport studies in cell monolayers, BEVs were labeled with FM 4-64 (Molecular Probes, Inc., Eugene, OR, United States) or by labeling RNA cargos by using the ExoGlow-RNA™ EV Labeling Kit (System Biosciences, Inc., Palo Alto, CA, United States) following the manufacturers’ recommendations. For transport studies in dual-chambers, BEVs were loaded with synthetic IRDye-labeled miR-34a as previously described (14 (link)).

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.