Cm h2dcfda

CM-H₂DCFDA (Thermo Scientific) and MitoSOX Red (Thermo Scientific) staining were used for detecting cytoplasmic and mitochondrial ROS levels, respectively. Cells were seeded in 24-well plates and cultured in normoxia or hypoxia for 24 h. Subsequently, cells were incubated with 10 μM CM-H₂DCFDA for 30 min or 5 μM MitoSOX Red for 10 min at 37°C, and fluorescence was captured by immunofluorescence microscope (Nikon, Tokyo, Japan) at 20 × magnification. Immunofluorescence density was quantified by Image J software (U. S. National Institutes of Health, MD, United States) and normalized by the siControl normoxia group.

Formation of reactive oxygen species (ROS) was evaluated in live hippocampal neurons using 2 μM CM-H2DCFDA (Life Technologies) as previously described [9 (link), 30 (link)]. Probe fluorescence was analyzed using NIH ImageJ software as described [30 (link)]. Briefly, hippocampal cultures were exposed to 500 nM AβOs for 2 h at 37 °C. hMSC-EVs (6.05 × 10⁷ or 1.82 × 10⁸ particles, as described in the “Results” section) were then added for 3 h and 30 min to the cultures. After this time, CM-H2DCFDA was loaded for 30 min and images were acquired on a Nikon Eclipse TE300 microscope. Nine microscopic fields were acquired using a × 20 objective per experimental condition in each of triplicate wells from three to four independent experiments using different hippocampal cultures and AβO preparations and were combined for analysis to allow quantitative estimates of changes in ROS levels. In some experiments (see the “Results” section), we used hMSC-EVs in which catalase had been inactivated by previous treatment at 37 °C with 1 mM aminotriazole for 30 min and washed twice with 20 mL PBS.

The total cellular peroxide concentration was determined by using the fluorescent probe CM-H₂DCFDA™ (#C6827, Themo Fisher Scientific, Loughborough, UK). After treatments, the medium was discarded. Fresh EGM2 medium containing 10 µM CM-H₂DCFDA was incubated at 37 °C for 30 min in the dark and analysed using a fluorescent microscope (Ex/Em: 485/530 nm, Nikon, Melville, NY, USA).