Jc 1 solution

HUVECs (1×10⁴ cells/mL) were treated with different concentration TF (0, 25 and 50 μM) for 24 h with or without 500 μM TBHP for 2 h. To detect the changes in mitochondrial membrane potential (MMP), the treated HUVECs were incubated in JC-1 solution (T3168, Invitrogen) at a concentration of 10 mg/L for 20 min at 37°C and then washed 3 times with PBS to remove excess JC-1 solution. Then, the stained samples images were immediately captured under a confocal microscope (Olympus, Tokyo, Japan). Lastly, the relative MMP was analysed with the ImageJ software. The relative MMP was calculated as a ratio of the mean fluorescence intensity of red fluorescence (excitation wavelength, 525 nm; emission wavelength, 590 nm) to green fluorescence (excitation wavelength, 490 nm; emission wavelength, 530 nm). To detect the changes in mitochondrial ROS generation, the treated HUVECs were stained in 5 nM MitoSOX Red Mitochondrial Superoxide Indicator solution (M36008, Invitrogen) for 30 min at 37°C. The fluorescence images were captured via a microscope (Olympus, Tokyo, Japan) under 510 nm excitation wavelength and 580 nm emission wavelength. Quantitation of mean fluorescence intensity by ImageJ was used to compare the ROS changes of the mitochondria.