Protease inhibitor cocktail tablets 1x

ASC-exosomes and adipose serum-deprived stem cells were lysed in 1XPBS containing protease inhibitor cocktail tablets 1X (Roche) for four times at 25 Hz for 10 s. Samples (60 μg per lane) were loaded in a native buffer and separated on a 15% (v/v) non-denaturing polyacrylamide gel for 3 h at 40 mA at 4 °C. The gel was stained for 45 min in 50 mM potassium phosphate (KH₂PO₄) pH 7.4 containing 275 μg/mL nitro blue tetrazolium (NBT) (ThermoFisher), 65 μg/mL riboflavin (Sigma-Aldrich) and 3.2 μL/mL tetra methyl ethylene diamine (TEMED) (Sigma-Aldrich) at room temperature in the dark. Gel was then illuminated for 15 min until sufficient contrast between achromatic zones (dismutase activity) and blue background was achieved and then scanned for documentation. ASC cells extract was used as positive control. An identical gel was stained with Coomassie brilliant blue to verify the equal amount of protein extract loaded among the different samples.

Exosomes and ASC were subjected to immunoblotting analysis using the following protocol: Proteins were extracted in RIPA buffer (Sigma-Aldrich) containing protease inhibitor cocktail tablets 1X (Roche), sonicated 10 min and then lysates were clarified by centrifugation at 8000× g for 10 min at 4 °C. Supernatants were harvested and protein content was determined by BCA assay (Sigma-Aldrich). Samples were denatured, separated on 10–20% polyacrylamide gels, transferred onto a PVDF (polyvinylidene difluoride) membrane and probed with antibodies against phospho-Akt (Ser473) (1:1000, Cell Signalling Technologies; #9271), and SOD1 (1:1000, NBP1-31204), followed by an appropriate HRP-conjugated secondary antibody against the primary antibody (Santa Cruz Biotechnology, sc-2005). ASC lysate was used as a positive control. The blots were then incubated with a chemiluminescent HRP substrate and detected with ChemiDoc MP imaging system (Bio-Rad).