Bicinchoninic acid protein quantification kit

Viruses were allowed to bind to established human cell clones cultured in 12 well plates (2.0 × 10⁵ cells/well). Briefly, cells were washed twice with PBS (+), inoculated with viruses at an m.o.i. of 10, and then incubated at 4 °C for 1 h to prevent endocytosis. The cells were then washed five times with ice-cold PBS (+) and lysed in PBS (−) containing 2% SDS. The protein concentration of the resulting cell lysates was measured using the bicinchoninic acid protein quantification kit (Thermo Fisher Scientific). Next, 10 μg of protein per lane was subjected to electrophoresis in a 10% SDS-polyacrylamide gel. The proteins were then transferred to PVDF membranes and blocked overnight at 4 °C in PBS (−) containing 5% non-fat milk and 0.1% Tween 20. The membrane was then exposed for 1 h at room temperature to the aforementioned rabbit polyclonal antivirus antibody (diluted 1:1000 in PBS (−) containing 5% non-fat milk and 0.1% Tween 20), followed by horseradish peroxidase-conjugated donkey anti-rabbit IgG (diluted 1:10,000 in PBS (−) containing 0.1% Tween 20; Jackson ImmunoResearch, West Grove, PA, USA). Between steps, the membranes were washed three times with PBS (−) containing 0.1% Tween 20. Reactive bands were visualized using a chemiluminescence system (Thermo Fisher Scientific) and Fuji XR film.

For extraction of extracellular vesicles (EVs), the hUCMSCs (passage 3) were incubated with an FBS-free medium for 48 h; subsequently, the culture supernatants were gathered. The gradient centrifugation was applied to isolate the extracellular vesicles. In brief, the centrifugal force of 1000×g, 10,000×g, and 120,000×g for 20 min, 30 min, and 90 min, respectively, at 4 °C was set at the ultracentrifuge (Beckman Coulter Optima L-80 XP) to centrifuge the culture supernatants. 100 μL of PBS was applied to resuspend the final extracellular vesicles pellet and stored at − 80 °C. The morphology of hUCMSCs-EVs was confirmed using transmission electron microscopy (TEM; HITACHI). Subsequently, a bicinchoninic acid protein quantification kit (Thermo Scientific, 23225) was employed to test the extracellular vesicles concentration. A light-scattering technique, nanoparticle tracking analysis (NTA; ZetaView PMX 110), was used to test the particle distribution and zeta potential. Surface characteristic proteins CD63, TSG101, and CD81 were further examined through western blot assay.

The protein expression of catalase, SOD1, and SOD2 in ASCs, spheroid-derived ASCs, and fibroblasts at low (2500 cells/cm²) and high (10000 cells/cm²) seeding densities was determined by western blot analysis. The cells were suspended in RIPA lysis buffer (Thermo Scientific, Rockford, IL). After centrifugation, the protein content was determined in the supernatants by a bicinchoninic acid protein quantification kit (Thermo Scientific). Protein samples from ASCs or fibroblasts was added to Laemmli sample buffer and boiled for 10 min. Subsequently, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto polyvinylidene difluoride membranes. Western blot was performed using anti-catalase, anti-SOD1, anti-SOD2 (all from Cell Signaling, Danvers, MA), and anti-GAPDH (Abcam, Cambridge, UK) antibodies. After overnight incubation with the primary antibodies and extensive washing, the membranes were further incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h. Then the blots were developed using an enhanced chemiluminescence detection system (Millipore, Billerica, MA). Blot images were taken using the UVP BioSpectrum^® Imaging System (Analytika Jena, Upland, CA) with VisionWorks Analysis Software (Analytika Jena).

Proteins were extracted from the cells using radioimmunoprecipitation assay buffer (Invitrogen), and then, the sample concentration was assessed using a bicinchoninic acid protein quantification kit (Thermo Fisher Scientific). Equal amounts of proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA, USA), transferred to polyvinylidene fluoride membranes (Millipore, Temecula, CA), blocked with 5% skim milk, and then incubated with the following primary antibodies: anti-YY1 (#46395, Cell Signaling Technology, Danvers, MA, USA, 1:1000), anti-PARP (#9542, Cell Signaling Technology, 1:1000), anti-Caspase-3 (#9662, Cell Signaling Technology, 1:1000), and anti-GAPDH (#5174, Cell Signaling Technology, 1:1000). Next, the membranes were incubated with secondary antibodies. Protein signals were observed using enhanced chemiluminescence (Bio-Rad), with GAPDH as an internal control (Olympus, Tokyo, Japan).

Reactive oxygen species assay kit was purchased from Us Everbright Inc (Suzhou, China). Bicinchoninic acid protein quantification kit was purchased from Thermofisher (Shanghai, China). Lipid peroxidation MDA assay kit, GSH assay kit, and GSH-Px assay kit were purchased from Beyotime Biotechnology (Shanghai, China). M-MuLV first strand cDNA synthesis kit was purchased from Gene Copoeia Inc (GuangZhou, China).