Exoglow protein ev labeling kit

Exosomes were isolated from cell culture media using ExoQuick-TC according to the manufacturer’s instructions (System Biosciences, Palo Alto, CA, USA) and stored at −80 °C until use.
To examine the uptake of exosomes by cells, exosomes were first labeled using the ExoGlow™-protein EV labeling kit (System Bioscience). The cells were treated with the labeled exosomes for 24 h, fixed using paraformaldehyde (Sigma, St. Louis, MO, USA), and imaged using a confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany).
Transfection of the miR-NC or miR-205-5p mimics into exosomes was performed using Exo-Fect™ according to the manufacturer’s protocol (System Biosciences).

Exosomes from ASCs were labeled using an ExoGlow-Protein EV Labeling Kit (System Biosciences; Palo Alto, CA) according to the manufacturer’s instructions. M1 macrophages were incubated in the labeled exosome suspension at a concentration of 30 μg/ml in Macrophage-SFM for the indicated times. The cells were then counterstained with Hoechst 33342 (Thermo Fisher Scientific) and examined by fluorescence microscopy (Olympus, Japan) and flow cytometry [18 (link)].

The isolated exosomes were labeled using an ExoGlow-Protein EV Labeling Kit (System Biosciences, Palo Alto, CA, USA). Differentiated 3T3-L1 cells were supplemented with labeled exosomes for 48 h, and exosome uptake was confirmed according to previously described methods [25 (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.

The isolated exosomes were labeled using the ExoGlow-Protein EV Labeling Kit (System Biosciences, Mountain View, CA, United States) and were incubated with the alpha mouse liver 12 (AML12) cells for 48 h. Following incubation, the cells were washed twice with PBS. The PBS was then aspirated, and the cells were fixed with 4% paraformaldehyde (PFA) for 15 min. Next, the cells were aspirated, washed twice with PBS, and permeabilized using 0.3% TritonX-100 for 1 h at 4°C. Nuclei were labeled using ProLong™ Gold Antifade Mountant with DAPI (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, United States). The slides were then covered with coverslips and observed under a fluorescence microscope (Olympus, Tokyo, Japan).