Pierce cell lysis buffer

A mixture of Pierce Cell Lysis Buffer (Thermo Fisher, USA) and Halt Protease Inhibitor Cocktail (Thermo Fisher, USA) was used to lyse exosome samples. The samples were then processed by electrophoresis and transferred to a polyvinylidene fluoride membrane (Bio-Rad, USA). Mouse anti-CD63 (sc-5275, 1 μg/mL, Santa Cruz, USA), mouse anti-HSP90 (ab13492, 1 μg/mL, Abcam, USA), and rabbit anti-TSG101 (ab30871, 1 μg/mL, Abcam, USA) were used as primary antibodies for incubating the membrane at 4 °C overnight. Goat anti-mouse IgG (ab97040, 0.05 μg/mL, Abcam, USA) and goat anti-rabbit IgG (ab97080, 0.05 μg/mL, Abcam, USA) were used as secondary antibodies. Protein abundances were analyzed with a ChemiDoc XRS+ system (Bio-Rad, USA).

The original plasma and isolated products in each experiment (sEVs, small exosome and exomere, exomere) of similar volumes (20 μl) were diluted to 200 μl using PBS. Pierce Cell Lysis Buffer (Thermo Fisher Scientific, USA) and Halt protease inhibitor cocktail (Thermo Fisher Scientific, USA) were used to lyse each sample. After processing by SDS–polyacrylamide gel electrophoresis, the lysates were transferred to a polyvinylidene fluoride membrane (Bio-Rad, USA). This membrane was then incubated with primary antibodies [mouse anti-CD63 (Santa Cruz Biotechnology, USA), mouse anti-HSP90, and rabbit anti-TSG101 (Abcam, UK)] for 12 hours at 4°C, followed by the incubation of appropriate horseradish peroxidase secondary antibody [goat anti-mouse immunoglobulin G (IgG) and goat anti-rabbit IgG (Abcam, UK)] for 1 hour at room temperature. Protein expression levels were finally characterized by ChemiDoc XRS+ (Bio-Rad, USA).

sEV samples isolated via FLOAT and sEV samples isolated via ultracentrifugation from equal volumes of cell-free urine were analyzed for sEV markers and circulating proteins. Twenty microliters of each sample was lysed in Pierce Cell Lysis Buffer (Thermo Fisher Scientific, USA) with Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, USA). Lysates were processed by SDS/PAGE and transferred to a polyvinylidene fluoride membrane (Bio-Rad, USA). Primary antibodies, including mouse anti-CD63 (Santa Cruz Biotechnology, USA), mouse anti-THP (Santa Cruz Biotechnology, USA), and rabbit anti-TSG101 (Abcam, USA), were separately used to incubate the membrane for 12 h at 4 °C. Appropriate horseradish peroxidase secondary antibodies, including goat anti-mouse IgG and goat anti-rabbit IgG (Abcam, USA), were used for a 1-h incubation at room temperature. ChemiDoc XRS+ (Bio-Rad, USA) was used to characterize protein expression levels.