Mitosox

The production of mitochondrial superoxide in mMSCs was measured using MitoSOX (Thermo Fisher Scientific). To measure mitochondrial membrane potential, tetramethylrhodamine, methyl ester, and perchlorate (TMRE; Thermo Fisher Scientific) dye was used. The mMSCs in each group were trypsinized and centrifuged at 1,200 rpm for 3 min. After washing with PBS twice, the cells were treated with either 10 μM MitoSOX in PBS or 200 nM TMRE in PBS at 37°C for 15 min. MitoSOX-positive or TMRE-positive cells were analyzed using flow cytometry (Sysmex, Kobe, Japan). flow cytometry data were analyzed using FCS Express 5 Flow research software (DeNovo Software, Los Angeles, CA, USA).

To measure the formation of mitochondrial O₂^•−, the mitochondrial superoxide of healthy-hMSCs or CKD-hMSCs was measured by means of MitoSOX™ (Thermo Fisher Scientific, Waltham, MA, USA), or TMRE (abcam, Cambridge, UK). These cells were trypsinized for 5 min and then centrifuged at 1200 rpm for 3 min, washed with PBS twice and then incubated with a 10 μM MitoSOX™ solution or 200 nM TMRE solution in PBS at 37 °C for 15 min. After that, the cells were washed more than 2 times with PBS. Next, we resuspended these cells in 500 μL of PBS, and then detected the signals with MitoSOX™ or TMRE by fluorescence-activated cell sorting (FACS; Sysmex, Kobe, Japan). Cell forward scatter levels were determined in MitoSOX™-positive cells, analyzed by means of the Flowing Software (DeNovo Software, Los Angeles, CA, USA).