CD63 exosome capture beads (ab239686, Abcam) were used to isolate EVs according to the manufacturer’s instruction. Briefly, 200 μl of plasma from healthy donors or patients with LUAD was centrifuged at 500g at room temperature for 10 min to remove residual cells. The supernatant was transferred to a new EP tube and centrifuged at 2000g at room temperature for 10 min to remove cell fragments. The supernatant was then transferred into a new EP tube and centrifuged at room temperature at 14,000g for 30 min to remove large vesicles. Last, the supernatant was transferred into a new EP tube and diluted with same volume of 1× PBS. Then, a volume of 200 μl of diluted plasma was aspirated into a new EP tube, and 100 μl of CD63 exosome capture beads was added and vortexed again. The sample was incubated overnight at room temperature in the dark. Following incubation, CD63-AF488 (NBP2-42225AF488, Novus Biologicals) and PD-L1–AF647 (ab209960, Abcam) fluorescent antibodies were added to the mixture at a volume ratio of 1:100 and incubated at 4°C for 2 hours in the dark. Stained EVs were sorted with a Navios cytometer (Beckman Coulter, CA, USA).
Ab239686
Manufactured by Abcam
Sourced in United States
Ab239686 is a laboratory equipment product manufactured by Abcam. It is designed to perform a specific function in a laboratory setting. No further details about its intended use or capabilities are provided to maintain an unbiased and factual approach.
Automatically generated - may contain errors
3 protocols using ab239686
1
CD63 Exosome Isolation and Characterization
2
Exosomal PD-L1 Detection Protocol
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Conditioned medium of BGC-823, MGC-803 and their corresponding LSD1 KO cells were incubated with CD63 exosome capture beads (ab239686, abcam, USA) in the dark overnight at room temperature. Then, the mixture was washed with 1 mL PBS twice, and the supernatant was discarded. After that, exosomes with beads were suspended in PE-conjugated anti-human PD-L1 antibody (1:5) (557924, BD, USA) for 40 min at 4℃. PE-conjugated IgG (556650, BD, USA) was set as negative control, then washed with PBS. Finally, the exosomes were analyzed by flow cytometry (BD, USA) after resuspension with 500 μL staining buffer. The whole process was protected from exposure to light.
3
Exosome Characterization by Flow Cytometry
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The hBMMSC-Exos were characterized by their surface markers CD63 and CD81 using flow cytometry. First, exosomes were captured with CD63-conjugated capture beads (ab239686, abcam, USA) and labeled with phycoerythrin (PE)-conjugated CD81 antibody (130-118-481, Miltenyi, USA) after serum blockage. CD63 and CD81 positive exosomes were quantified using a flow cytometer (Novocyte 2000, Agilent, USA) against isotype control.
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