Cytation3 multifunctional microplate reader

Cardiomyocyte mitochondrial damage was assessed by using the mitochondrial membrane potential assay kit with JC-1, according to the protocols provided by the manufacturer (Beyotime, Hangzhou, China). JC-1 is a marker of mitochondrial activity, which is most widely applied for detecting mitochondrial depolarization occurring in the early stages of apoptosis. JC-1 aggregates in the mitochondria and gives off a red fluorescence in healthy cells. In apoptotic cells, JC-1 fails to aggregate in the mitochondria as a result of altered mitochondrial transmembrane potential and remains in the cytoplasm in its monomer form, which exhibits green fluorescence.
Briefly, the cells were incubated with 0.5 ml JC-1 staining solution for 20 min at 37 °C, and the fluorescent signals were observed by a fluorescence microscopy and measured by a CYTATION3 multifunctional microplate reader (Biotek). The red fluorescent signals were excited at 525 nm and detected at 590 nm, and the green fluorescence was excited at 490 nm and detected at 530 nm. The data were shown as the ratio of red signals versus green signals.

Intracellular ATP levels were assessed through the firefly luciferase-based ATP assay kit (Beyotime), according to manufacturer’s instruction. Briefly, the cultured neonatal cardiomyocytes were lysated using ATP lysis buffer. After centrifugation to remove cell debris, 20 μl of supernatant was added to 100 μl of ATP detection solution at its working dilution. Luminance (RLU) was measured using a CYTATION3 multifunctional microplate reader (Biotek) with an integration time of 10 s per well and the concentration of ATP was calculated according to the standard curve. ATP levels were normalized to the protein levels.

Mitochondria-mediated reactive oxygen species (ROS) generation was detected with the mitochondrial superoxide indicator MitoSOX-Red (Invitrogen). Briefly, the cells were washed twice in PBS and incubated with 5 mol/l of MitoSOX-Red working solution for 10 min at 37 °C, protected from light. Then, the cells were washed gently three times with warm buffer, and the fluorescent signals were measured using a CYTATION3 multifunctional microplate reader (Biotek) with the excitation wavelength at 510 nm and emission wavelength at 580 nm, or analyzed using FACS Verse flow cytometer (BD).