摘要
目的建立以刃天青为氧化还原指示剂测定mtDNA被完全清除的大鼠胶质瘤C6细胞(C6ρ0)增殖及活力的方法,并对其有效性进行验证。方法取对数生长期的C6ρ0细胞,采用刃天青法比较不同细胞数量、刃天青浓度、孵育时间对荧光强度的影响,优化测定C6ρ0细胞活力的条件。比较MTT法和刃天青法测定C6ρ0和C6细胞活力的灵敏性,并以天然化合物熊果酸对C6ρ0和C6细胞活力的影响对该方法进行验证。结果 C6ρ0细胞数为0~30 000/孔,25μg/mL刃天青培养基避光孵育1~4 h,细胞数量与荧光强度呈良好线性关系,灵敏性高于MTT法。熊果酸0~50μmol/L对C6ρ0和C6细胞具有增殖抑制作用,且增殖抑制率差异有统计学意义(P<0.05),IC50分别为20.82、14.62μg/mL。结论刃天青法灵敏、简单、安全无毒、经济便宜、重复性好,特别适用于生长代谢缓慢的细胞系,可用于其细胞增殖测定、活力分析及化合物细胞毒的高通量筛选。
Objective To establish a method for determining the cell viability of mitochondrial DNA(mtDNA)-depleted C6 cells(C6ρ0) using resazurin as the indicator of redox. Methods We performed a systematic analysis to determine whether C6ρ0 cells were able to reduce resazurin and whether there was a linear correlation between resazurin reduction and cell viability. The conditions of the resazurin assay were optimized by evaluating the fluorescence intensities in cell cultures with different cell numbers, varying concentrations of resazurin, and after different incubation time. This resazurin assay was assessed for its sensitivity in detecting the viability of C6ρ0 and C6 cells in comparison with MTT assay. Results In resazurin assay, an optimal linear relationship between the cell number and the fluorescent intensity was maintained when the cell density was 0~30 000 per well and the cells were incubated in a 96-well plate with 25 μg/mL resazurin in the dark for 1 to 4 h at 37 ℃ with 5% CO2. In the cytotoxicity test of ursolic acid using resazurin assay, ursolic acid significantly inhibited the proliferation of C6ρ0 and C6 glioma cells(P<0.05) with IC50 values of 20.82 μg/mL and 14.62 μg/mL, respectively. Conclusion The resazurin assay allows sensitive, nontoxic and cost-effective determination of the cell number of mtDNA-depleted cells, and can also be used for cell proliferation and viability assessment, with high-throughput screening of cytotoxic compounds.
作者
靳有才
张一鸣
李忌
王洪伦
白波
JIN Youcai;ZHANG Yiming;LI Ji;WANG Honglun;BAI Bo(Key Laboratory of Tibetan Medicine Research, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining, Qinghai Province, 810008;Department of Environmental Science, College of Chemistry and Chemical Engineering, Qinghai Normal University, Xining, Qinghai Province, 810008;University of Chinese Academy of Sciences, Beijing, 100049;Center for Mitochondria and Healthy Aging, College of Life Sciences, Yantai University, Yantai, Shandong Province, 264005, China)
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2019年第12期1136-1141,共6页
Journal of Third Military Medical University
基金
中国科学院战略性先导科技专项(A类)(XDA12040320)~~
