Mouse anti human cd63

When cell confluence reached 70–80%, hAECs were washed with phosphate-buffered saline (PBS) and incubated with DMEM containing 10% exosome-free FBS for 48 hours. The conditioned medium was collected, successively centrifuged at 300 × g for 10 minutes, 2000 × g for 10 minutes and 10,000 × g for 30 minutes to remove dead cells and cell fragments, and finally passed through a 0.22 um filter (Millipore, Billerica, USA). Next, the filtrate was centrifuged at 100,000 × g for 2 hours, washed twice with 500 μL PBS and recentrifuged at 100,000 × g for 2 hours to collect pure exosomes. All procedures were conducted at 4°C. Exosomes suspended in 100 μL PBS were stored at −80°C for further experiments. The size distribution of hAECs-Exos was analysed by dynamic light scattering (DLS) with a Malvern Nanosizer (Malvern Instruments, UK). The morphology of hAECs-Exos was observed by transmission electron microscope (TEM) (Hitachi H-7650; Hitachi, Japan) as described previously in detail [10 (link)]. The surface markers (CD63, TSG101) of hAECs-Exos were analysed by flow cytometry (FCM) with the primary antibodies mouse anti-human CD63 (Santa Cruz Biotechnology, USA) and rabbit anti-human TSG101 (ProteinTech, USA) as described previously [11 (link)]. Finally, the protein level of hAECs-Exos was quantified with a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, USA) for subsequent studies.

The samples from RBC-derived exosomes, BSA-PEG-micelles, and the peak fractions for the BSA-PEG-lipid-exosomes from size-exclusion chromatography were analyzed by Western blotting. The protein concentrations of all samples were estimated using the BCA assay kit (KeyGEN Biotech). Then, equal amounts of protein were separated on a 10% SDS-PAGE and transferred to the PVDF membrane (Millipore) using a high quality wet protein transfer system (L00686C eBLOT L1, Genscript, Nanjing, China). The membranes were then blocked with 5% skim milk powder in TBST buffer for 1 h at RT. The blots were then incubated overnight at 4℃ with the primary antibodies including mouse anti-BSA (1:2000, Proteintech), mouse anti-human CD63 (1:1000, Santa Cruz), mouse anti-human CD9 (1:1000, Proteintech), or mouse anti-human Alix (1:1000, Santa Cruz). Subsequently, the blots were incubated with HRP-conjugated goat anti-mouse IgG antibodies (1:5000, Cell signaling) for 1 h at RT. Then, the blots were developed using the Enhanced chemiluminescence detection kit (BL520A, Biosharp).