Fig. 5

Effects of food derived micro/nano-particles on cell plasma membrane potential (indicated with green fluorescence of DiBAC₄(3)) and mitochondrial superoxide (indicated with red fluorescence of MitoSOX) of murine oral mucosal macrophages. Cells were loaded with 3 μM MitoSOX Red for 10 min and then 2.5 μM DiBAC₄(3) for 15 min at 37 °C. For AAPH treated cells (6.4 μM, incubated for 120 min), they were loaded with fluorescence dyes prior to the addition of AAPH and micro/nano-particles. a: Fluorescent micrographs (red & green fluorescence, taken by a Leica DMI3000 B Inverted Microscope, excitation at 515~560 nm and 450~490 nm, respectively) of loaded cells, merged with software LAS (version 4.2.0) wherein UF-MNPs/cell ratio = 3500/1,700/1 and 140/1; SEC-NPs/cell ratio = 3500/1,700/1 and 140/1. b: V _mem-dependent fluorescence traces (1 data-point/10 min for 120 min, excitation/emission at 493/516 nm for DiBAC₄(3)) of loaded macrophages in a 96-well microplate were measured simultaneously by a fluorometric plate reader (Flexstation3, Molecular Devices, USA). c: the diagram shows the impacts of UF-MNPs, SEC-NPs and AAPH on the mitochondrial superoxide level of macrophages, indicated by MitoSOX Red fluorescence (excitation/emission at 510/580 nm). *: P < 0.05 v.s. Control, **: P < 0.01 v.s. Control; #: P < 0.05 v.s. AAPH, ##: P < 0.01 v.s. AAPH. The oral macrophages partially presented green-yellowish color due to the overlap between two fluorescent probes in cell compartments or cell groups. Among normal macrophages, cells with higher level of mitochondrial superoxide (brighter red fluorescence) tend to have hyperpolarized membranes (weaker green fluorescence) which carry more intensively the negative charge. AAPH induced peroxyl radicals eliminated the DiBAC₄(3) fluorescence as well as decreased MitoSOX Red fluorescence, indicating the hyperpolarization of cell membrane and down-regulated mitochondrial oxygen respiratory