Pierce bca protein assay kit

Cells (washed three times with PBS) and the tissues were lysed in a RIPA lysis buffer (CW2333, CWBIO, China) with protease inhibitor (CW2200, CWBIO, China) and phosphatase inhibitor cocktail (CW2383, CWBIO, China) for 45 min. Protein concentrations of cell lysates and tissue lysates were determined using a Pierce BCA Protein Assay Kit (CW0014, CWBIO, China) according to the manufacturer's instructions. Total protein (30-50 μg) for each sample was separated by the SDS–PAGE and transferred to polyvinylidene difluoride (PVDF, Millipore, IPVH00010) membranes. The membranes were blocked with 5% nonfat milk at room temperature for 2 h and then incubated with primary antibodies overnight at 4°C (for all antibodies, see Supplementary Table2). After washing, the membranes were incubated with a 1 : 8,000 dilution of goat anti-mouse or anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (CW0102 and CW0103, CWBIO, China) at room temperature for 1.5 h. Protein bands were visualized using an Immobilon Western Chemiluminescent HRP Substrate (WBKLS0500, Millipore) via an automatic chemiluminescence imaging analysis system (Tanon-5200, USA). ImageJ (NIH, National Institutes of Health, USA) was used to analyse these images.

A total of 1 × 10⁶ cells were treated with 200 μL of RIPA lysis buffer (CWBIO, Beijing, China, CW2333S) and 2 μL of protease inhibitor PMSF (Solarbio, Shanghai, China, P0100). Total protein was quantified using the Pierce BCA Protein Assay Kit (CWBIO, Beijing, China, CW0014S) and denatured at 100 °C for 10 min. Equal amounts of the lysates were fractionated by 12% SDS-PAGE (GenScript, Nanjing, China, M01210C). Proteins were transferred onto PVDF membranes (Solarbio, Shanghai, China, YA1701) using a Semi-Dry Electrophoretic Transfer Cell (Bio-Rad, California, USA, Trans-Blot^® SD). After the membranes were blocked overnight with 5% skim milk (Bio-Rad, California, USA, 1706404) and washed with TBST three times, they were probed with antibodies against GFP (CWBIO, Beijing, China, CW0087M, 1:1000) and β-actin (CWBIO, Beijing, China, CW0096M, 1:500). Goat anti-rabbit antibody (CWBIO, Beijing, China, CW0107S, 1:1000) and goat anti-mouse antibody (CWBIO, Beijing, China, CW0102S, 1:2000) were used as secondary antibodies. Uncropped blots can be found in the Source data file.

sEVs were isolated from cell culture medium or plasma through differential centrifugation as previously described.^[9] Plasma samples were diluted in PBS before centrifugation. Briefly, after removal of cells and other debris by centrifugation at 300 g for 10 min, 2000 g for 10 min, 10000 g for 30 min, cell supernatant was centrifuged at 110 000 × g for 70 min (all of these steps were performed at 4°C). The sEVs were collected and resuspended in PBS or FBS‐free medium. A NanoSight N300 instrument (Malvern, UK) was used to analyze the distribution of sEVs sizes. sEVs quantification was conducted by measuring the protein content of sEVs pellets with the Pierce BCA Protein Assay Kit (CWBio, China).