Accuri cflow plus software

Cells were stained with the oxidant-sensitive fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA; cat. no. 35845; Sigma-Aldrich; Merck KGaA) for 15 min and were washed with PBS. 2′,7′-Dichlorofluorescein (DCF) was generated from DCFH-DA in the presence of peroxide. The fluorescence of DCF was detected using Accuri C6 flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) with an excitation wavelength of 485 nm. Data analysis was performed using Accuri CFlow Plus software (version 1.0; BD Biosciences).

The JC-1 probe can emit green fluorescence at low membrane potentials and red fluorescence at increased potentials. MitoProbe™ JC-1 Assay kit (cat. no. M34152; Invitrogen; Thermo Fisher Scientific, Inc.) was used to determine MMP, according to the manufacturer's protocol. Briefly, the cells were stained with JC-1 for 20 min at 37°C and were then loaded onto an Accuri C6 flow cytometer (BD Biosciences). The ratio of red/green fluorescence was calculated and a decrease in the ratio indicated loss of MMP. Data analysis was performed with Accuri CFlow Plus software (version 1.0; BD Biosciences).

The Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit (cat. no. V13241; Invitrogen; Thermo Fisher Scientific, Inc.) was used to detect apoptosis. Annexin V-FITC⁺/PI⁻ indicated early apoptotic cells, whereas late apoptotic cells were represented as Annexin V-FITC⁺/PI⁺. According to the manufacturer's protocol, following treatment, cells in a 24-well plate (1×10⁵ cells/well) were stained with Annexin V-FITC and PI for 10 min at room temperature in the dark, after which fluorescence intensity was analyzed using Accuri C6 flow cytometry with Accuri CFlow Plus software version 1.0 (BD Biosciences).