Photobiomodulation at Defined Wavelengths Regulates Mitochondrial Membrane Potential and Redox Balance in Skin Fibroblasts

Figure 4

Effect of three different frequencies of the 645 nm LED light as a pre-exposure treatment on H₂O₂-induced oxidative stress. (a) Exposure protocol: to analyze the effect of the frequency, we performed a single 20 s. exposure using the 645 nm LED light at three different frequencies (50, 100, and 150 Hz) on fibroblast cultures. After 24 hr, cells were stained with MitoTracker, fixed, and processed for analysis by cell-based HCS. (b) The graph shows the relative MitoTracker intensity. Results are expressed as a percentage of the nonexposed group (100%; horizontal dotted line); n = 4. (c) Representative images of the nonexposed (Ctrl) and exposed cells. Scale bar: 100 µm. (d) Exposure protocol: to analyze the possible protective effect against oxidative stress, we exposed the fibroblast cultures for 20 s, inducing oxidative stress through the H₂O₂ (40 µM) treatment after 24 hr. After a further 24 hr, cells were stained with MitoTracker and CellROX dyes, fixed, stained with hoechst, and processed for the analysis by cell-based HCS. (e–g) Graphs show the effect on mitochondrial potential as the relative intensity of the MitoTracker staining (e), on ROS production as the relative intensity of CellROX staining (f), and cell death as relative percentage of condensed nuclei (g); n = 4. All the data are expressed as a percentage of the vehicle group not treated with H₂O₂ or exposed to the LED light (100%), whereas the control group treated with H₂O₂ but not exposed to the LED light is indicated by the white bar. (h) Representative HCS images of cells not treated with H₂O₂ or exposed to the LED light (vehicle); treated with H₂O₂ but not exposed to the LED light, and treated with H₂O₂ and exposed to the LED light at the three different frequencies (50, 100 and 150 Hz). Scale bar: 50 µm. Statistical analysis. Data are presented as average values ± SEM. One-way ANOVA followed by Dunnett’s postdoc was used to compare groups. Asterisks represent statistically significant differences between the indicated group and the nonexposed control (b) or the H₂O₂-treated/non-exposed control (e–g) (; ; ; ).

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