Exoglow membrane ev labeling kit

The exosome size was determined using a dynamic light scattering system (Otsuka ELS-Z, Japan). Exosomes isolated from the plasma of WT and CrebH^−/− mice were quantified using an ExoELISA-ULTRA assay kit (SBI System Biosciences) according to the manufacturer’s guidelines [28 (link)]. Exosomal markers were estimated by immunoblotting using antibodies against TSG101 (Abcam), CD9 (Abcam), and Histone H3 (Sigma-Aldrich). Exosomes were labeled using an ExoGlow-Membrane EV labeling kit (SBI System Biosciences) following the manufacturer’s instructions. Labeled exosomes were cocultured with bEnd.3 cell lines for the indicated time. Images were obtained using fluorescence microscopy (Olympus, Tokyo, Japan). Exosomal proteins were quantified using Bradford protein assay and bEnd.3 cells were treated with exosomes (30 µg/mL protein concentration contained in exosome) for 24 h.

EVs were stained with ExoGlow-membrane EV labeling kit (System Biosciences, Pala Alto, CA, USA) according to the manufacturer’s instructions. After labeling, the EVs were mixed with 10 ml of PBS and the EVs were isolated again by centrifugation at 100,000× g for 1 h. Cells were seeded onto a microscope cover glass. After culturing overnight, the culture media containing the fluorescence-labelled EVs were treated for 3~24 h. The cells were fixed with 4% paraformaldehyde in PBS. After washing, the nuclei were stained using 4,6-diamidino-2-phenylindole and mounted in Vectashield^® (Vector Laboratories Inc., Burlingame, CA, USA). Images were observed under the Nikon Eclipse E400 microscope (Nikon Instruments Inc., Melville, NY, USA) using a Nikon Digital site DS-U2 (Nikon Instruments Inc.), and analyzed using NIS element F (version 4.6, Nikon Instruments Inc.).

To monitor the uptake of EV^Avo by peritoneal macrophages, EV^Avo was labelled with Exo‐GLOW Membrane EV Labeling Kit (System Biosciences) per the manufacturer's instructions.
⁴⁶ (link) Briefly, EV^Avo (300 μg) was mixed with the Exoglow fluorescent dye and incubated at RT for 30 min in the dark. We used 10 K MWCO buffer exchange tubes and centrifuged samples at 12000 g for 10 min to remove free unlabeled dye. Labelled EV^Avo (2 or 10 μg/mL) was then added to macrophages and their uptake by the cells at different time points was visualized by fluorescence microscopy (Zeiss Axio Observer, White Plains, NY).

Urinary exosomes from responder, non-responder, and healthy controls were extracted as described previously (n = 5 for each group). To fluorescently label, 100 µg of exosomes were incubated for 30 min at room temperature with labeling dye (465/635 nm excitation/emission) provided for the ExoGlow-Membrane EV Labeling kit (System Biosciences, Palo Alto, CA, USA). The unlabeled dye was removed using the PD SpinTrap G-25 buffer exchange column (GE Healthcare, Chicago, IL, USA). Afterwards, 10 µg of labelled urinary exosomes were added to 24-well plates with mesangial, endothelial, or epithelial tubular cells. Cell internalization was analyzed at 3, 6, and 18 h by immunofluorescence microscopy.

EVs were labeled with ExoGlow membrane EV labeling kit (Cat# EXOGM600A-1, System Biosciences, Palo Alto, CA) according to the manufacturer’s instructions. EV concentration and size distribution were analyzed via single-molecule tracking using the ONI Nanoimager (Oxford Nanoimaging; ONI) with a 488-nm wavelength laser at an exposure of 10 ms for 5000 captured frames.

EXOs were labeled with a ExoGlow-Membrane EV Labeling Kit (Cat #EXOGM600A-1, System Biosciences, Palo Alto, CA, USA) in which a labeling reaction buffer was prepared in a 1:6 dilution of labeling dye. Next, 14 µL of the reaction buffer was added to the EXOs, which were then incubated for 30 min at room temperature. To remove excess dye, we isolated the labeled EXOs by using the Invitrogen Total Exosome Isolation Reagent as described above. We plated 0.5 × 10⁴ of ECs and cardiomyocytes, and after 24 h of recovery, the cells were cultured in the presence of the labeled EXOs under standard conditions. The EVOS M700 was used to capture the internalization of gelatin-EXOs and LAMA2-EXOs into the cells over time.