2′,7′-dichlorofluorescein diacetate (DCF) measurement of reactive oxygen species (ROS)

Cardiomyocytes were loaded with 1 μM DCFH-DA (Beyotime) for 20 min to measure ROS⁴⁴. Non-fluorescent DCFH could be oxidized to fluorescent DCF. Detecting the fluorescence of DCF can quantify the level of ROS in myocytes. All experiments were performed on the Nikon A1 confocal microscope (Nikon Instruments) 100× oil objective (N.A. = 1.49) using the 488 nm excitation wavelength. A total of 30 μM Yoda1 (Maybridge) was immediately added before camera shooting. For testing the blocking effect of Rac1, DCF images were taken and compared before and after application of 50 μM Rac1 inhibitor (APExBIO) together with 30 μM Yoda1. For testing the blocking effect of NOX2, 3 μM membrane permeable gp91ds-tat (Anaspec) were pre-incubated 30 min before the application of Yoda1, and the images were taken and compared before and after the application of Yoda1. Cells were imaged at low laser intensity and the 488 nm laser was shut off during the drug delivery process to ensure that the same position was taken twice without causing artifactual signal changes of DCF.

For measuring stretch-induced ROS, cardiomyocytes were loaded with 1 μM DCF for 20 min as described above. The attached cardiomyocytes were given an 8% of axil stretch of the cell length and recorded DCF fluorescence intensity. DCF loaded cells were imaged using confocal line-scanning microscopy. As DCF can produce artifactual signal amplification upon continuous light exposure, cardiomyocytes were imaged at low laser intensity. Meanwhile, the attached cells were first recorded for the baseline fluorescence without stretch, then followed by stretch-induced fluorescence change. The baseline fluorescence was subtracted from the stretch-induced fluorescence change to avoid artifactual fluorescence change caused by illumination of DCF itself. Confocal line-scans were taken from the interior of the myocytes in the stretched region of the cells.