Acid phosphatase assay

ADMSCs were pretreated with Fragaria-derived EPDENs (0.5–1–2 μg/mL) for 24 h followed by 400 µM hydrogen peroxide (H₂O₂) exposure for 24 h. The H₂O₂ concentration that caused the inhibition of 50% cell viability (IC50) was determined from the dose–response curve ranging from 0 to 1000 µM H₂O₂. Cell viability was evaluated using the acid phosphatase assay (Sigma-Aldrich), as previously described [12 (link)]. Optical density was read at 405 nm using a microplate reader. Data were reported as cell survival with respect to untreated cells (set = 100%).
ROS were detected by the 2′,7′-dichlorofluorescin (DCFH) method. Briefly, ADMSCs were pretreated with EPDENs (0.5 µg/mL) for 24 h followed by 600 μM H₂O₂ exposure for 1 h. The H₂O₂ concentration that significantly increased ROS production was determined from a dose–response curve ranging from 0 to 800 µM H₂O₂. ADMSCs were washed and then incubated with 10 μM 5- and 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA; Thermo Fisher Scientific, Monza, Italy) for 5 min at 37 °C. The fluorescent signal of DCF obtained from the conversion of 2′,7′-dichlorofluorescin-diacetate (DCFH-DA) by the intracellular ROS produced was read at 485–535 nm using a microplate reader (Tecan Infinite F200pro). The results were expressed as the mean of the relative fluorescence units (RFUs).

Cell viability was evaluated by the acid phosphatase assay (Sigma-Aldrich). HUVEC cells were seeded in 96-well, flat-bottom tissue culture plates coated with 0.2% gelatin in water at a density of 1 × 10⁴ cells/well in Endothelial Cell Growth Medium plus Endothelial Cell Growth Supplement. After 24 h, cells were treated with 2–6 μg/w of OS-derived EVs (pH 6.5 or 7.4) or PBS (control), in the presence of Endothelial Cell Growth Medium + 1% FDE. At 48 h, the cells were washed and incubated at 37 °C with 100 μL of buffer containing 0.1 M sodium acetate pH 5.0, 0.1% Triton X-100, and 5 mM p-nitrophenyl phosphate. After 2 h, the reaction was stopped with the addition of 10 μL of 1 N NaOH, and color development was assayed at 405 nm using a microplate reader (Tecan Infinite F200pro, Männedorf, Switzerland). Data are reported as cell survival in respect to untreated cells (set = 1). The experiment was performed three times in triplicate.

Male Wistar rats were fasted for 48 h before euthanasia for lysosomal isolation. Lysosomes were isolated using the lysosome isolation kit (Sigma-Aldrich) in a Optiprep density gradient, following step by step the instructions of the technical bulletin. In all cases 6 franctions were isolated after ultracentrifugation of the liver tissue and the acid phosphatase assay (sigma-aldrich) was performed to access the fractions positive for lysosomes. From each sample 2 fractions were isolated that were enriched in acid phosphatase. Samples of the homogenate and and lysosomal fractions, containing 100 ug of protein, were denatured in sample buffer and runned in SDS-PAGE.