Bx50 fla fluorescence microscope

Intracellular ROS levels were detected by the oxidative conversion of DCFH-DA (cell-permeable) to dichlorofluorescein (DCF, fluorescent). Following being cultured in 24-well plates (7×10⁶), H9c2 cells were treated differently and washed with PBS twice, into which serum-free medium containing 10 µM DCFH-DA solution was thereafter added to incubate the cells for 60 min at 37°C. They were then washed with PBS three times, DCF fluorescence in which was detected over the whole visual field by BX50-FLA fluorescence microscope (Olympus Corporation) connected with an imaging system. In addition, the mean fluorescence intensity (MFI) of four randomly selected fields was determined with ImageJ software (version, 1.41o; National Institutes of Health, Bethesda, MD, USA). ROS levels were represented by MFI of DCF.

MMP was detected by Rh123, a cell-permeable, fluorescent cationic dye preferentially entering mitochondria on the basis of a highly negative MMP. MMP depolarization leads to Rh123 loss from mitochondria, thus decreasing intracellular fluorescence. H9c2 cells were herein cultured in 24-well plates (7×10⁶) and treated differently. Following addition of Rh123 (100 mg/l) into the culture medium, the cells were further incubated at 37°C for 45 min and observed under BX50-FLA fluorescence microscope (Olympus Corporation) connected with an imaging system. The MFI of Rh123 from four randomly selected fields, which was analyzed using ImageJ software, represented the level of MMP. The experiments were repeated three times.

The apoptosis of H9c2 cells was evaluated by Hoechst 33258 staining. Following different treatments, the cells were fixed for 10 min with 4% paraformaldehyde in phosphate-buffered saline (PBS), then washed by PBS three times, stained for 5 min by 5 mg/l Hoechst 33258, rinsed again by PBS and observed under BX50-FLA fluorescence microscope (Olympus Corporation, Tokyo, Japan). The viable cells emitted uniform blue fluorescence and presented normal nuclear sizes, but the apoptotic ones had fractured, condensed or distorted nuclei.