Elucidating mechanism cellular uptake removal protein
SEA Block Blocking buffer was from Thermo Scientific.
Di I-labeled acetylated LDL was from Harbor Bioproducts. Complete protease inhibitors and phosphatase inhibitors were from Roche.
Protein amounts were quantified with BCA protein assay kit (Pierce) in each vesicle preparation.
Isolated exosomes were in indicated experiments labeled with PKH67, PKH26 or cellvue midklaret fluorescent labeling kit according to the manufacturer's protocol (Sigma).
Background: Exosome vesicles can transfer molecular information previously shown to stimulate tumor development; however, the mechanism of exosome uptake is unknown.
Results: Mammalian cells internalize exosomes through lipid raft-mediated endocytosis negatively regulated by caveolin-1.
Each vesicle preparation was stored at 4 °C and used within 5 days after isolation.
All experiments were performed in Zeiss LSM 710 confocal scanning equipment using excitation wavelengths of 405, 488, 546, 633 nm, and a Plan-Apochromat 20×/0.8M27 objective and a C-Apochromat 63X/1.20W korr M27 glycerol immersion objective.
Transmission electron microscopy was performed to validate the presence and purity of intact exosomes and analyzed for size using nanoparticle tracking analysis (NTA).
We find that exosomes induce the phosphorylation of several downstream targets known to associate with lipid rafts as signaling and sorting platforms, such as extracellular signal-regulated kinase-1/2 (ERK1/2) and heat shock protein 27 (HSP27).