Fluorescence activated cell sorting (facs)

Autophagy staining (Sigma-Aldrich) was used to observe autophagosomes in cultured MSCs. The cells were incubated with autophagy stain (10 μM) for 30 min. After washing with PBS twice, these cells were fixed in 500 μL 4% PFA (paraformaldehyde) for 1 h. These cells were measured for autophagosomes by using FACS (Sysmex, Kobe, Japan). Autophagosome-positive cells were analyzed by using Flowing Software (De Novo Software, Los Angles, CA, USA).

Generation of mitochondrial O₂^•− in hMSCs was assayed using MitoSOX (Thermo Fisher Scientific). The cells in each group were trypsinized and centrifuged at 600 g for 5 min. The samples were then washed with PBS and incubated with 10 μM MitoSOX solution in phosphate-buffered saline (PBS) at 37 °C for 15 min. Next, the cells were resuspended in 500 μL PBS, and the total number of cells labeled using MitoSOX was measured via FACS (Sysmex, Kobe, Japan). MitoSOX-positive cells (number of events: 10⁴) were identified and analyzed using Flowing Software (DeNovo Software, Los Angeles, CA, USA).

To quantify the formation of mitochondrial O₂^•−, we used TMRE (Abcam, Cambridge, UK) to measure generation of mitochondrial superoxide in hMSCs subjected to oxidative stress. For this, hMSCs were trypsinized for 5 min, centrifuged at 1200 rpm for 3 min, washed with PBS twice, and incubated with 200 nM TMRE solution in PBS at 37 °C for 15 min. The cells were then washed two times with PBS and resuspended in 500 μL PBS. Following this, TMRE signaling was detected by fluorescence-activated cell sorting (FACS; Sysmex, Kobe, Japan). Cellular forward-scatter levels for TMRE-positive cells (number of events: 10⁴ cells) were analyzed using Flowing Software (DeNovo Software, Los Angeles, CA, USA).