Dmi fluorescent microscope

Intracellular ROS production was assessed by using the specific probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Beyotime Institute of Biotechnology), according to the manufacturer's instructions. Intracellular ROS oxidize DCFH-DA, yielding the fluorescent compound 2′,7′-dichlorofluorescein (DCF) and measurement of the DCF fluorescence intensity is representative of the amount of ROS in the cells. H9c2 cells were cultured in 6-well plates, then exposed to 30 mM sodium azide for 24 h, with or without pretreatment with 1 µM Mdivi-1 for 3 h. Subsequently, cells were treated with DCFH-DA (10 µM) dissolved in serum-free DMEM (1:1,000) for 20 min at 37°C and then washed three times with serum-free DMEM. The cells were then observed for green fluorescence (excitation 488 nm, emission 525 nm) with a Leica DMI fluorescent microscope (Leica Microsystems GmbH) and analyzed with ImageJ v1.32 J software (National Institutes of Health). Fluorescence intensities of ROS were presented in arbitrary units (a.u).

H9c2 cells, with or without 1 µM Mdivi-1 pretreatment for 3 h, were incubated with 30 mM sodium azide for 24 h. In order to distinguish between programmed or non-apoptotic cell death, nuclei were stained with DAPI. Briefly, cells were washed twice with PBS and then fixed with 4% paraformaldehyde for 30 min at room temperature. Following three washes, fixed cells were stained with DAPI (1:5,000; dilution with PBS) for 5–10 min. Cells were then washed with PBS and fluorescence images were captured with a Leica DMI fluorescent microscope (Leica Microsystems GmbH, Wetzlar, Germany). The experiments were repeated at least three times.