Platelet-poor plasma was prepared from 6 ml of venous blood and stored in 0.5 ml aliquots at − 80 °C as previously described26 (link) and EDE were enriched as previously published26 (link). Briefly, after depletion of platelets, EDE exosomes were enriched by sequential immunoprecipitation with two biotinylated monoclonal antibodies to CD31 (MEM-05, Thermo Fisher Scientific) and then CD146 (Novus Biologicals, Littleton, CO, USA) prior to lysis of exosomes for quantification of cargo proteins via ELISA. Sequential rounds of immunoprecipitation aimed to enhance selectivity. We used Nanosight NS300 instrument (Malvern Instruments, Malvern, UK) in combination with an EV membrane label, ExoGlow (System Biosciences, Palo Alto, CA) to confirm that the particle sizes of total exosomal extracts were within range for exosomes (30–220 nm).
Exo glow
Manufactured by System Biosciences
Sourced in United Kingdom, United States
Exo-Glow is a bioluminescent labeling reagent that can be used to label extracellular vesicles (EVs) or exosomes. It provides a simple and fast way to visualize and track the biodistribution of EVs in vitro and in vivo.
Automatically generated - may contain errors
Lab products found in correlation
Exoquick tc, by System Biosciences (2 mentions)
Nanosight ns300 instrument, by Malvern Panalytical (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Glass bottom 96 well plate, by MatTek (1 mentions)
Volocity software, by PerkinElmer (1 mentions)
Ultraview vox, by Nikon (1 mentions)
Sp5 aobs confocal laser scanning microscope, by Leica (1 mentions)
Dmi6000 inverted epifluorescence microscope, by Leica (1 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (1 mentions)
Huvecs, by Lonza (1 mentions)
Opera phenix high content screening system, by PerkinElmer (1 mentions)
11 protocols using exo glow
1
Isolation and Characterization of Endothelial Exosomes
2
Exosome Tracking in Vivo using Exo-Glow
3
Exosome Quantification Using NanoSight NS300
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NanoSight NS300, with the Blue488 laser and sCMOS camera, was used in combination with ExoGlow (System Biosciences, Palo Alto, CA) to quantify the total EV population (5 videos, 30 s each) and confirm that size distribution is within range for exosomes (30–220 nm) (Supplemental Fig. S2 ). After capture, the videos were analyzed using the NTA 3.3 Dev Build 3.3.104 software, with a detection threshold of 4 and screen gain of 10. As expected, the non-fluorescent NTA captured non EV vesicles with a wider size range (peak ~ 100 nm), while fluorescent NTA with EV membrane dye demonstrated a peak concentration of around 30-50 nm and lower overall concentration of particles, likely due to increased specificity for EVs.
4
Fluorescent Labeling of EV Membrane
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For fluorescent labeling of the EV membrane, Exo-Glow (System Bioscience, Palo Alto, CA), acridine orange nucleic acid-selective fluorescent dye was added to the purified EV according to the manufacturer's instructions. After EV membranes were fluorescently labeled, ultracentrifugation was performed at 100,000 × g for 60 min to remove the unlabeled dye. The labeled EVs were then added to HUVECs. After 4–8 h of incubation, the cells were gently washed with 1 × PBS and analyzed by flow cytometry or fluorescence microscopy.
5
In vivo Exosome Tracking Protocol
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To track exosomes in vivo, Exo‐Glow (System Biosciences) was used to label purified exosomes prior to intravitreal injection, according to the manufacturer's instructions. Briefly, 3 × 108 exosomes were suspended in 500 μl of sPBS and incubated with Exo‐Green labeling solution for 10 minutes at 37°C followed by 30 minutes on ice. Labeled exosomes were isolated by treatment with Exoquick‐TC (System Biosciences), centrifugation for 30 minutes at 14,000g, washed three times with sPBS before being resuspended in 500 μl of sPBS and kept on ice until intravitreal injection on the same day.
6
Tracking Extracellular Vesicle Uptake Dynamics
7
Fluorescent Labeling of EV mRNA for Cellular Uptake
8
Exosome Uptake in Endothelial Cells
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PF-derived exosomes were labeled using Exo-Glow based on carboxyfluorescein succinimidyl diacetate ester (CFSE) chemistry (System Biosciences) according to the manufacturer’s recommendations. Human umbilical vein ECs (HUVECs) (Lonza) were seeded at a density of 5 × 104 cells/well on a 24-well plate coverslip, and 10 μg/ml of labeled PF-derived exosomes were added to target ECs in culture for 24 hr at 37°C. Cells were washed twice with PBS and fixed with 4% buffered PFA (Sigma) in PBS for 20 min at RT. Nuclei were stained by DAPI staining while actin filaments were labeled using Rhodamine Phalloidin (Thermo Fisher Scientific). To assess the uptake of exosomes by ECs, confocal images were acquired with a Leica SP5-AOBS confocal laser scanning microscope attached to a Leica DM I6000 inverted epifluorescence microscope. All images were collected using a 63× NA 1.4 oil immersion lens objective. The excitation signals for Exo-Glow and Rhodamine Phalloidin were 494 and 540 nm, respectively. The fluorescence emitted from the cells was recorded at 521 nm for Exo-Glow and 565 nm for Rhodamine Phalloidin. In all cases, z-stack images were obtained covering the entire cell volume. Three-dimensional reconstruction of the confocal image z stacks confirmed the cytoplasmatic localization of internalized exosomes.
9
Exosomal Protein Fluorescent Labeling
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The exosomal proteins were labeled fluorescent green with Exo‐Glow (System Biosciences) according to the manufacturer's protocol. Further consecutive visualization with fluorescence microscopy was performed for 24 hours.
10
Labeling and Tracking MBV Cargo in Cells
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MBV nucleic acid cargo was labeled using Exo-Glow (System Biosciences), according to the manufacturer’s instructions. Briefly, 500 μl of resuspended MBVs was labeled with Exo-Glow and incubated at 37°C for 10 min. ExoQuick-TC (100 μl) was added to stop the reaction, and samples were placed on ice for 30 min. Samples were then centrifuged for 10 min at 14,000g. The supernatant was removed, and the pellet was resuspended with 500 μl of 1× PBS; 50 μl of this MBV suspension was added to C2C12 in culture. The cells were cultured for 4 hours, and the transfer of the MBV cargo to the cells was determined by imaging using a 100× objective and Axio Observer Z1 microscope.
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