Caspases represent a family of cysteine proteases that are common downstream effectors of apoptosis (Chen et al., 2001). After irradiation, the culture medium was removed and adherent cells were trypsinized. Melanoma cells and melanocytes were pelleted by centrifugation at 1800 rpm for 10 min and incubated with 1 μg of specific Anti-caspase 3 PE antibody (Santa Cruz, USA) and 10 μL of Triton X-100 (0.1%) for 1 h at 4 °C. The cells were UK-371804 solubility dmso then resuspended in FACS Flow buffer. The samples were analyzed for fluorescence (FL-1H detector) on a Becton Dickinson FACScan
flow cytometer using Cell Quest software. This experiment was performed 6 h after thermal neutron irradiation. The caspase-3 inhibitor zDEVD-fmk (Becton Dickinson, USA) was prepared as stock solution in 100% DMSO (100 mM). see more Final concentration in serum-free medium was 1 mM for zDEVD-fmk. Cells were incubated with caspase inhibitor 1 h before addition of BPA. Control wells were incubated with corresponding DMSO concentration. The values are expressed as the mean ± standard deviation (s.d.). The data were analyzed using one-way analysis of variance (ANOVA), and significant mean differences were determined using multiple comparisons by the Tukey–Kramer test at the p < 0.05 level. Significant differences between the control and treated groups are indicated by *** p < 0.001, ** p < 0.01, and * p < 0.05. Melanocytes treated with BNCT showed low levels of cell death. The IC50 value was 34.4 mg/mL, which corresponds to 1.8 mg/mL 10B (Fig. 1). The cellular viability (IC50 value) of the irradiated control did not show any significant difference compared to the control group. After BNCT treatment, the melanocytes exhibited an increase in free radical production, and this increase was greater only when higher BPA concentrations were used (Fig. 2). However, the increase in free radical production in the highest BPA concentration used was approximately only 1.5 times higher than that of the control group. The lower BPA concentrations did not show significant differences. The irradiated control also did not exhibit Axenfeld syndrome any differences compared to the control group. The normal melanocytes were photographed for morphological analysis after BNCT treatment. None of the BPA concentrations induced morphological changes. The presence of apoptotic bodies, debris formation and cytoskeleton disarray was also not detected (Fig. 3). Only the highest BPA concentration showed a slight decrease in confluence, which is consistent with the free radical production observed when using this concentration. The cells of the irradiated control presented insignificant alterations and little cell damage. After BNCT, the extracellular matrix of normal melanocytes and melanoma cells was analyzed by Sirus Red staining. The extracellular matrix of melanoma cells treated with BNCT showed dramatic changes, as evidenced by a decrease in soluble collagen synthesis (Fig. 4).