Enzyme labeled secondary antibody

According to the instructions, the levels of TNF-α, IL-1β, and IL-6 were detected using an Enzyme Immunoassay Kit (Boster, Inc., Wuhan, China). Briefly, when the macrophages reached 70% fusion, the cell supernatant was collected, the antigen was added and kept at 4°C overnight, and then enzyme-labeled secondary antibody (Abcam Inc., Cambridge, MA, USA) was added and incubated at room temperature for 1 h. Finally, the optical density of each sample at 450 nm was measured, and the levels of TNF-α, IL-1β, and IL-6 were calculated according to the regression equation of the standard curve.

ELISA kits (Shanghai Enzyme-linked Biotechnology Co., Ltd. Shanghai, China) were performed to detect interleukin-6 (IL-6), IL-1β and tumor necrosis factor alpha (TNF-α). IL-6 (ab100713, Abcam, Cambridge, U.K.), IL-1β (ab46052, Abcam, Cambridge, U.K.), and TNF-α (ab34674, Abcam, Cambridge, U.K.) were diluted by antibodies to 1–10 μg/ml, with 0.1 ml added to each well for incubation at 4°C overnight, after which the samples were washed three times the following day. The above reaction wells were added with certain amount of dilute supernatant (0.1 ml) and incubation was carried out at 37°C for 1 h and washed. Simultaneously, the blank, negative, and positive control wells were prepared in the reaction well, and 0.1 ml freshly diluted enzyme labeled secondary antibody (Abcam Inc., Cambridge, MA, U.S.A.) was added for incubation at 37°C for 35–40 min and washed, followed by washing with ddH₂O (PER 018-1, Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China). Each reaction well was added with temporarily prepared TMB substrate solution (EL0001, InnoReagents, Zhejiang, China) at 37°C for 10–30 min, and 50 μl stop buffer was added to terminate the development. Finally, the optical density (OD) of each well was determined at the wavelength of 450 nm within 20 min. Each experiment was conducted three times.

Firstly, a certain amount of RIPA lysis buffer (Biyuntian, China) was added to the collected exosome pellets or cells or tumor tissues to extract the total protein, and then the total protein of protein concentration was quantitatively analyzed using the BCA kit (Solarbio, China). Subsequently, the loading buffer was added to the sample and boiled for 5 min. After the protein was denatured, it was added to a 12% polyacrylamide gel for electrophoresis separation (constant voltage for 200 V). After that, the separated protein was transferred to PVDF membrane (Millipore, USA) using an electro-membrane transfer instrument. Then, the PVDF membrane was immersed in 5% skimmed milk for 2 h at room temperature, and then incubated with the diluted primary antibody and enzyme-labeled secondary antibody (Abcam, UK) in sequence. Finally, ECL luminescent solution (BBI, China) was added, and the gel imaging analysis system was used to image the protein.
Dilution ratio of the antibodies used in this experiment was as followed: Primary antibodies: anti-CD63 (ab193349), 1:1000; anti-CD9 (ab58989), 1:2000; anti-Bcl-2 (ab117115), 1:5000; anti-Calnexin (ab241154). 1:1000; Secondary antibody: goat anti-mouse (ab182017), 1:2000.