Methods
Materials and Instruments
5-ALA, fetal bovine serum (FBS), 2′,7′-dichlorodihydrofluorescein diacetate, and MTT (Sigma) were used. Our study also utilized the following materials and instruments: enzyme labeling instrument produced by Bio-Rad Biomedical Products (Shanghai) Co., Ltd., Dulbecco's modified Eagle medium (DMEM) by Shanghai Yuanpei Biotechnology Co., Ltd., semiconductor laser by Beijing Bingshou Technology Co., Ltd., annexin V-fluorescein isothiocyanate/propidium iodide apoptosis detection kit by BD Co., flow cytometer by BD Co.; ribonuclease A by Beyotime Biotechnology Co., Ltd., human OSCC cell line SCC25 from ATCC that was cultured at 37 °C, 5% CO2.
Photodynamic Therapy Parameters
The treatment setting was: 87 mW/cm2 power, 635 nm irradiation wavelength, 10.4 J/cm2 energy density.
Detection of Protoporphyrin IX in SCC25 Cells
SCC25 cells were divided into the observation group and the blank control group. Different concentrations of 5-ALA (5, 10, 25, 50, 100 and 150 mg/L) and the observation group cells were co-incubated for different lengths of time (2, 4, 6, 12 and 24 h).
The growth of protoporphyrin IX in SCC25 cells was observed by flow cytometry after treatment with 5-ALA. SCC25 cells in logarithmic growth phase were evenly spread in a 12-well plate at 8 × 104 cells per well, and 1 mL DMEM containing 15% FBS was added to each well and cultured for 24 h. After removal of the culture medium containing serum, the supernatant was removed; after wash with sterile phosphate buffer for two times, DMEM (without fetal bovine serum) was used to prepare 5-ALA. The concentrations were set as 5 mg/L (the 5 mg/L group), 10 mg/L (the 10 mg/L group), 2 5 mg/L (the 25 mg/L group), 50 mg/L (the 50 mg/L group), 100 mg/L (the 100 mg/L group), 150 mg/L (the 150 mg/L group) and 0 mg/L (the blank control group). 5-ALA and SCC25 cells were incubated for 2, 4, 8, 12 and 24 h, and the concentration of protoporphyrin IX in cells was detected by flow cytometry. The intracellular average fluorescence intensity was analyzed by C Flow Plus 1.0.264.15 software by BD Co. (USA).
MTT was Used to Detect the Cell Activity
SCC25 cells were divided into the 5-ALA alone group (100 mg/L), the laser irradiation alone group, the 5-ALA combined with laser irradiation group (5-ALA concentration was set from low to high as 5, 10, 25, 50 and 100 mg/L), and the blank control group (0 mg/L 5-ALA).
Activity of SCC25 cells was detected by MTT after treatment with 5-ALA combined with laser irradiation. SCC25 cells in the logarithmic growth phase were evenly spread in a 96-well plate at 6 × 103 cells per well, and 100 μL of DMEM containing 15% FBS was added to each well and cultured for 24 h. The supernatant was removed. After wash with sterile phosphate buffer for two times, the Dulbecco's modified Eagle medium (without fetal bovine serum) was used to prepare 5-ALA. After co-incubation of 5-ALA and SCC25 for 4, 8 and 12 h, the cells were irradiated by laser. After the supernatant was discarded, 100 μL DMEM containing 15% FBS was added to each well. After 24 h of culture, 20 μL MTT solution was added to each well to stop the culture. The cells were incubated in the dark for 4 h, and then the supernatant was removed. Then, 150 μL dimethyl sulfoxide was added, and mixed for 8 min. Absorbance was read at 490 nm, and the cell survival rate was calculated as follows:
Apoptosis was Detected by Flow Cytometry
SCC25 cells were divided into the 5-ALA alone group (100 mg/L), the laser irradiation alone group, the 5-ALA combined with laser irradiation group (5-ALA concentration was set from low to high as 5, 10, 25, 50 and 100 mg/L), and the blank control group (0 mg/L 5-ALA).
After co-incubation of 5-ALA and SCC25 cells for 12 h, the cells were irradiated by laser, and then cultured in DMEM with 15% FBS for 24 h. Apoptosis of SCC25 cells was induced by 5-ALA combined with laser irradiation, and apoptosis was detected by annexin V-fluorescein isothiocyanate/propidium iodide double staining. Five μL annexin V-fluorescein isothiocyanate/propidium iodide were added. After 5 min, the apoptosis of SCC25 cells was detected by flow cytometry. The experiment was done in triplicate.
The Effect of 5-ALA on SCC25 Cell Cycle was Detected by Flow Cytometry
Cells in the logarithmic growth phase were placed in a 6-well plate at a density of 5 × 105 mL− 1 and cultured at 37 °C for 24 h. The cells were divided in two groups: the 5-ALA (100 mg/L) combined with laser (2 h) group and the blank control group. After 24 h, the cells were collected and washed twice with sterile phosphate buffer twice. The single cell suspension was fixed with 70% ethanol, the temperature was set at 4 °C, and the fixed solution was washed with sterile phosphate buffer. Then, 100 μ L ribonuclease A was added at 37 °C water bath for 30 min, and mixed with 400 μL propidium iodide solution in the dark at 4 °C. After 30 min, the results were detected and recorded by flow cytometry. The above experiments were repeated 3 times in each group to ensure the accuracy of the data.
Statistical Analysis
Data were expressed as x̅ ± s and analyzed by IBM Microsoft SPSS 21.0 software. Repeated measurement analysis of variance was used for overall comparison, t-test was used for pairwise comparison of inter-group and intra-group data. Categorical data was expressed as rate (%), and chi-square χ 2 test was used for comparison. A P < 0.05 represented significant statistical difference.
BMC Oral Health. 2020;20(258) © 2020 BioMed Central, Ltd.
