In order to determine the optimal hypericin concentration or irradiation dose for PDT treatment, we performed MTT assay to analyze the proliferation of SP2/0 cells [29 (link)]. Briefly, SP2/0 cells at a final density of 5 × 104 cells/ml (100 μl/well), were incubated with hypericin at a concentration gradient from 0.001 to 10 μM for 16 hours in the dark. The cell plates were illuminated with different light dose of 0, 2.82, 5.64, 8.46, 11.28 and 14.10 J/cm2, and then incubated for 24 hours in the dark. To obtain the IC50, SP2/0 cells were treated with indicated concentrations of hypericin (0, 0.0125, 0.025, 0.05 and 0.1 μM) for 16 hours in the dark. Subsequently, the cells were illuminated at a light dose of 11.28 J/cm2 for 24 hours in the dark.
SP2/0 cells without any treatment were set as controls. The MTT assay was performed for SP2/0 cells at each condition according to the manufacturer’s instructions. In brief, 10 μl of MTT (5 mg/ml; Sigma) was added in each well for 4 hours at 37°C. After removal of the medium, the MTT crystals were dissolved with 100 μl DMSO for 15 min. The absorbance at 550 nm was measured by a Multiskan Spectrum 1500 (Thermo Electron Corporation, USA). The inhibition rate of cell proliferation was calculated as follows: (A550control − A550treated)/A550control × 100% and the IC50 value was obtained by Logit method [30 (link)].
SP2/0 cells without any treatment were set as controls. The MTT assay was performed for SP2/0 cells at each condition according to the manufacturer’s instructions. In brief, 10 μl of MTT (5 mg/ml; Sigma) was added in each well for 4 hours at 37°C. After removal of the medium, the MTT crystals were dissolved with 100 μl DMSO for 15 min. The absorbance at 550 nm was measured by a Multiskan Spectrum 1500 (Thermo Electron Corporation, USA). The inhibition rate of cell proliferation was calculated as follows: (A550control − A550treated)/A550control × 100% and the IC50 value was obtained by Logit method [30 (link)].