Diphenyl 1 pyrenylphosphine dppp

Cells were stained with 5 µM of a fluorescent probe diphenyl-1-pyrenylphosphine (DPPP; Molecular Probes) as described (Morita et al. 2016 (link)) and analyzed using an Olympus FV1200 laser scanning microscope equipped with the FV10-ASW viewer 4.2 software. Mouse skin tissue was analyzed by immunohistochemistry using an antibody to 4-hydroxy-2-nonenal (4-HNE) (Cosmo Bio Co., Tokyo, Japan).

For measurement of antioxidant activities in the zebrafish, the embryos (3 dpf) were transferred to a 12-well plate. The embryos were treated with DCFH-DA (20 μg/mL), and incubated in the dark room for 1 h at room temperature for ROS measurement. The embryos were washed using fresh medium and anesthetized using Ethyl 3-aminobenzoate methanesulfonate (Sigma-Aldrich, St. Luis, MO, USA) before observation. We placed each anesthetized larva on the slide glass, and fluorescence values were measured using a fluorescence microscope. The differences in the fluorescence intensities of the groups were noted by keeping the value of the control group fixed as the index for the comparisons. Lipid peroxidation was measured by treatment with diphenyl-1-pyrenylphosphine (DPPP, Thermo Scientific, USA). Similarly, embryos in the well plates were treated with DPPP (25 μg/mL) and incubated. Washing and anesthetizing was carried out, as mentioned above. Cell death was measured by treatment with acridine orange (Sigma-Aldrich, St. Louis, MO, USA), (7 μg/mL). The observation of zebrafish was conducted using Cool SNAP-Pro color digital camera (Olympus, Japan) according to Kim et al [13 (link)]. The fluorescence intensities of the zebrafish larvae were quantified using Image J (Version 1.51) program.

Suspensions of 10⁷C. glabrata stationary-phase cells/ml were exposed to caspofungin (0.25 and 1 μg/ml) or hydrogen peroxide (5 and 10 mM) and incubated with or without 50 μM diphenyl-1-pyrenylphosphine (DPPP) (ThermoFisher) at 37°C (68 (link), 69 (link)). The fluorescence intensity was measured every hour using a Tecan infinite 200 Pro plate reader with a 340/380 nm excitation filter. Data are shown in arbitrary units of fluorescence intensity after 2 and 6 hours of caspofungin exposure.