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

Figure 3

Mitochondrial analysis on fibroblasts exposed to 645 nm LED light. (a) Exposure protocol: using the red-emitting LED (645 nm), we performed a single 20 s exposure to analyze the potential mitochondrial changes after 24, 48, and 72 hr. At each time point, cells were stained with MitoTracker dye, fixed, and processed for analysis. (b) The graph shows the relative MitoTracker intensity, measured at each time point after exposure. Results are expressed as percentage of the nonexposed group (100%; horizontal dotted line) for each time point; n = 3. (c) Representative images of the nonexposed (Ctrl) and exposed cells (645 nm) 24, 48, and 72 hr after the exposure. Bar: 50 µm. (d) Exposure protocol: to analyze mitochondrial fusion, we performed a single 20 s. exposure using two different LED light sources (645 and 900 nm). After 24 hr, cells were stained with MitoTracker, fixed, processed for immunocytochemistry for MFN2 (Mitofusin 2) marker identification, and processed for analysis. (e) Graph shows the ratio between the number of MFN2 spots and the total mitochondrial net volume per single cell, identified by the MitoTracker staining; n = 10. (f) Representative images of a cultured fibroblast, nonexposed (Ctrl) or exposed to the LED light (645 or 900 nm). Images were acquired by confocal microscopy as z-stacks and analyzed by IMARIS software. MitoTracker staining was initially used to create a mask to measure and isolate the mitochondrial net volume, then the “spot detection” algorithm was used to identify and count all MFN2 spots inside the identified volume. Scale bar: 5 µm. Statistical analysis. Data are presented as average values ± SEM. One-way ANOVA followed by Dunnett’s postdoc was used to compare groups in the same exposure protocol. Asterisks represent statistically significant differences between the indicated group and its corresponding nonexposed control (; ; ; ).

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