HUVECs were cultured in DMEM with the addition of NaH2PO4 for the HP (3 mM; pH=7.4) group or without NaH2PO4 for the no phosphorus (NP) group for 48 h at 37°C in a humidified atmosphere with 5% CO2 for exosome isolation. NaH2PO4 (MilliporeSigma) was used to increase the concentration of phosphorus in the medium. Before extraction of exosomes from HUVECs, the cells were cultured with medium supplemented with 10% exosome-depleted FBS for 48 h at 37°C with 5% CO2 (C38010100; Shanghai VivaCell Biosciences, Ltd.).
Exosomes were concentrated using an ExoQuick-TC Exosome Precipitation Solution kit (EXOTC10A; System Biosciences, LLC). This method has been widely used before and has been proven to collect exosomes effectively (21 (link)-23 (link)). Briefly, the supernatant was centrifuged at room temperature as follows: 300 × g for 10 min, 2,000 × g for 30 min and 10,000 × g for 30 min. An Amicon Ultra15 Centrifugal Filter Unit (100 kDa; MilliporeSigma) was used to concentrate the supernatant. The ultrafiltration liquid and exosome isolation reagents were mixed at a 5:1 ratio and incubated at 4°C for ~16 h. Finally, the mixture was centrifuged at room temperature at 1,500 × g for 30 min, and the exosome pellets were resuspended in 200 µl PBS. Protein quantification of exosomes was performed using a BCA kit (Beyotime Institute of Biotechnology).
Exosomes were concentrated using an ExoQuick-TC Exosome Precipitation Solution kit (EXOTC10A; System Biosciences, LLC). This method has been widely used before and has been proven to collect exosomes effectively (21 (link)-23 (link)). Briefly, the supernatant was centrifuged at room temperature as follows: 300 × g for 10 min, 2,000 × g for 30 min and 10,000 × g for 30 min. An Amicon Ultra15 Centrifugal Filter Unit (100 kDa; MilliporeSigma) was used to concentrate the supernatant. The ultrafiltration liquid and exosome isolation reagents were mixed at a 5:1 ratio and incubated at 4°C for ~16 h. Finally, the mixture was centrifuged at room temperature at 1,500 × g for 30 min, and the exosome pellets were resuspended in 200 µl PBS. Protein quantification of exosomes was performed using a BCA kit (Beyotime Institute of Biotechnology).
