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Int. J. Mol. Sci. 2018, 19, 1107 7 of 17 Figure 3. Toxic effects of 160 μM 4 min MB and 8 min laser exposure on Detroit 562 cells. Laser exposure alone ensured 95.8% ± 2.2% cell viability, which was not significantly different from the control (p = 0.76 with Mann Whitney test); 160 μM 4 min MB ensured 46.1% ± 0.9% cell viability, which was significantly different from the control (****: p < 10−4 with Mann Whitney test), the combined MB + 660 nm laser exposure allowed 4.9% ± 0.5% cell viability, which was significantly different from the MB exposure and from the 660 nm laser exposure alone (****: p < 10−4 with Mann Whitney test). 2.3. PDT Effects on Clonogenic Survival The main interest of the current study is not the analysis of cell viability, which might be a transient reduction of MTT-assay measured mitochondrial and cytoplasmic enzymes [44] but to estimate the tumor reproduction capacity of HNSCC tumor cells after the above-detailed optimized MB and laser treatment conditions. In this regard, the capacity of both SCC-25 and Detroit 562 cells was investigated for producing the numbers of progeny in clonogenic assays. After completion of treatments, 2000 cells/treatment were seeded in 75cm2 culture dishes in 15 mL serum-supplemented culture medium for 2 weeks followed by counting the Gentian Violet-stained visible colonies (approximately 50 cells in each colony) (Figure 4). In the culture of SCC-25 cells 173 ± 13 colonies have grown from the plated 2000 cells in conditions of scattered light (control), 160 μM MB alone did not induce significant change in the number of colonies, whereas 160 μM MB (4 min) + 8 min laser treatment high significantly reduced the number of surviving colonies to 38 ± 8 (p < 0.01, by Mann-Whitney test, n = 4; four biological repeats) (Figure 4A). In the culture of Detroit 562 cells, 466 ± 44 colonies have grown from the plated 2000 cells in conditions of scattered light (control), 160 μM MB alone did not induce significant change in the number of colonies, whereas 160 μM MB (4 min) + 8 min laser treatment high significantly reduced the number of the surviving colonies to 19 ± 26 (p < 0.01, by Mann-Whitney test, n = 4; four biological repeats) (Figure 4B).PDF Image | Photodynamic Low Level Laser Squamous Cell Carcinoma
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