Cellrox reagent

H₂O₂ generation was measured using Amplex Red and horseradish peroxidase (HRP) as previously described (Vázquez-Medina et al., 2016 (link)). Briefly, cells were incubated with 50 μM Amplex Red (Thermo Fisher, MA) and 2.5 U/ml HRP (Sigma) for 30 min at 37°C. The medium was collected, and absorbance was measured at 572 nm. At the end of the experiments, the cells were dissociated from the dishes. Protein content was measured by the BCA gold assay (Thermo Scientific, MA) and results were normalized to protein content. Intracellular oxidant generation was monitored by fluorescence microscopy using CellROX reagent (Thermo Scientific, MA). Cells loaded with 5 μM CellROX and NucBlue (Thermo Fisher, MA) were incubated for 30 min at 37°C. Cells were rinsed three times and imaged using an inverted fluorescence microscope (Zeiss Axio Observer) fitted with a 20× objective and Zen software. Fluorescence intensity in five fields per sample was quantified using ImageJ (NIH) and normalized to cell number.

Tailfin transections were performed on zebrafish larvae at 4dpf. The CellROX Reagent (Thermo fisher) was added to the fish water at a final concentration of 5 μM and incubated for 30 minutes at 32°C. Fish water with CellROX was removed and the larvae were rinsed 3 times with clean fish water. Live imaging of larva was performed using a Zeiss Axiovert wide field microscope system, images were taken every 10 minutes after tail fin transection. The background subtracted integrated fluorescence intensity of CellROX in the tailfin region was then quantified using Zen software.

ROS levels were measured using the Cell Rox Reagent (Thermo Fisher Scientific, catalog C10444) in HD- and BT-MSCs at the basal level or after 5 days of iron treatment, according to the manufacturer’s instructions. Cell viability was analyzed by using 7AAD (BD Biosciences, catalog 559925) prior to analysis.

MSC metabolic activity was measured by a colorimetric assay based on the reduction of XTT [2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide] by nicotinamide adenine dinucleotide (NADH) (ThermoFisher, Manhattan, NY, USA). The reagent is reduced by NADH produced during mitochondrial metabolism which results in a color change of the XTT reagent detectable by a spectrophotometer. The concentration of the reagent is measured by absorbance measured at a wavelength of 450–500 nm. This assay was performed on MSC that were in suspension for 30 and 120 min in perfusion fluid or culture medium and on attached MSC. In addition, oxidative stress of MSC was measured using CellRox reagent (ThermoFisher) according to the manufacturer's manual. CellRox is oxidized by reactive oxygen species (ROS) and emits a fluorescent signal that is measured by flow cytometry.

Cardiomyocytes derived from iPSCs (iPSC-CMs) were treated with 500 μM H₂O₂ for 2 h, and subsequently incubated with iMSC-EVs or HA-iMSC-EVs in serum-free DMEM for 24 h. Next, the CellROX® Reagent (Thermo Fisher Scientific) was mixed with serum-free DMEM to a final concentration of 5 μM and added to the culture for 30 min. Following staining, the cells were fixed in 4% paraformaldehyde (Fujifilm Wako Chemicals, Richmond, VA, USA) for 10 min and washed three times with DPBS. Nuclei and cell bodies were counterstained with NucBlue^™ Fixed Cell stain or CellTracker^™ (Invitrogen), respectively. After this process, all samples were observed using a Nikon Eclipse Ti2-U (Nikon, Tokyo, Japan), and the percentage of ROS-positive cells was analyzed based on nuclear intensity.