Mitosox kit

An Annexin V-FITC/PI (BD, 556,419) kit was used to determine the levels of apoptosis and necrosis in HEI-OC1 cells. In brief, HEI-OC1 cells were trypsinized and centrifuged at 300 ×g for 5 min. After washing 3 times with cold PBS, cells were incubated with FITC Annexin V in a buffer containing propidium iodide and analyzed by flow cytometry (FACSCanto, BD, San Jose, CA, United States).
A Mito-SOX kit (Thermo Fisher, M36008) was employed to measure the oxidative stress level of HEI-OC1 cells. Cells were trypsinized and collected by centrifugation at 300 g for 5 min. The cells were then resuspended in Mito-SOX solution for 10 min, washed three times in PBS, and analyzed by flow cytometry.

Mitochondrial ROS formation after treatment was assessed using the MitoSOX kit (ThermoFisher, Watlham, MA, USA) modified as described by Kauffman et al. [43 (link)]. First, 10⁵ cells were resuspended in 500 μL of PBS, and then 1 μL of a 2.5 μM MitoSOX dye solution in DMSO was added. After 20 min of incubation at 37 °C, cells were centrifuged and resuspended in 500 μL of fresh PBS. Subsequently, the cells were analysed by flow cytometry.

Mitochondrial ROS was assessed using a MitoSOX kit (Thermo Fisher Scientific, M36008, USA) following manufacturer's protocols. After transfection and treatment, MitoSOX was added to the H9c2 cells and incubated at 37° C for 20 min. The relative fluorescence intensity was calculated in different groups.

Actinomycin D, antimicyn A (AA), carbonyl cyanide m-chlorophenylhydrazone (CCCP), 2’,7’-dichlorodihydrofluorescein diacetate (H₂DCFDA), digitonin, dimethylsulfoxide (DMSO), rhodamine 123 (Rh123), and Nile red were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Invitrogen (Grand Island, NY, USA). Annexin-V FITC, the MitoSOX kit, propidium iodide (PI), and the TUNEL kit were obtained from Invitrogen (Eugene, OR, USA). All of the other reagents were of analytical grade.

Mitochondrial ROS of HRECs were assessed using a MitoSOX kit (M36005; Invitrogen). Mito-Track Green (C1048; Beyotime Biotechnology) staining was used for mitochondrial localization. The fluorescence intensity was analyzed using confocal laser scanning microscopy.

Mitochondrial superoxide contents were measured using the MitoSOX kit (Invitrogen) according to the manufacturer's instructions. Briefly, HUVECs were treated with indicated concentrations of BA in the presence or absence of Mito-TEMPOL (2 μM) and NAC (3 mM) for 12 h. After that, the cells were harvested and incubated with 5 μM MitoSOX reagent to incubate cells for 10 min in the dark at 37°C. The cell's mean fluorescence intensity (MFI) was then detected using the BD Accuri C6 Plus flow cytometry.

Mitochondria ROS were detected by MitoSOX kit (#M36008, Invitrogen). H9C2 cells were exposed to 0 or 16 Gy X-ray the next day. MCELNs (0 or 10 μg/mL) were added to the culture medium before radiation exposure. After 48 h of culture, cells were harvested with 0.25% trypsin without EDTA and washed with PBS three times. Then, cell pellets were resuspended with 1 mL of 5 μM MitoSOX™ reagent working solution and incubated for 10 min at 37°C in the dark. After washing with PBS three times, cells were analyzed using a flow cytometer (FACS Canto II, Becton Dickinson, New Jersey, U.S.).