Following transfection, A-549 cells were seeded in 25 cm2 culture flasks at a density of 20×104/well in DMEM, and harvested when they reached 70% confluence. Then, cells were stained with 5 µl Annexin V-FITC (BD Biosciences) for 15 min and 5 µl propidium iodide (PI) for another 5 min at room temperature. Cell cycle was determined using a FACScanto Accuri C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and the data was analyzed using CFlow Plus 1.0.264.15 software (BD Biosciences).
Cflow plus 1
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CFlow Plus 1.0.264.15 is a software application designed for lab equipment. It provides core functionality for managing and analyzing data related to the lab equipment's operations.
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3 protocols using cflow plus 1
1
Apoptosis Assay with A-549 Cells
2
Cell Cycle and Apoptosis Analysis of DFSCs
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DFSCs transfected with si-Nkd2 or si-NC for 72 h were used for cell cycle distribution and apoptosis analyses. For the cell cycle distribution assay, 1×106 treated cells were fixed in 70% pre-chilled ethanol overnight at -20°C. Subsequently, the cells were labeled with Forevergen Cell Cycle kits (Forevergen; Guangzhou Yongnuo Biotechnology Co., Ltd., Guangzhou, China) according to the manufacturer's protocol. The Annexin V/propidium iodide apoptosis assay was performed using the PE Annexin V Apoptosis Detection kit I (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's protocols. All samples were analyzed using an Accuri C6 Flow Cytometer and CFlow Plus 1.0.264.15 Software (BD Biosciences).
3
Protoporphyrin IX Accumulation in SCC25 Cells
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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).
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).
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