三种试剂用于两种肿瘤细胞活性检测的比较研究
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摘要
目的通过对三种试剂用于两种肿瘤细胞活性检测性能的综合对比,提供有理论依据的实践参考。方法分别以贴壁细胞Hela和悬浮细胞SU-DHL-2为研究对象,对CCK-8、alamarBlue和CellTiter-Glo三种常用试剂的最佳条件进行探索。结果对于两种细胞,三种试剂在一定条件下均有较好的线性区间,Pearson相关系数r>0.99;CCK-8用于检测Hela的细胞数目为8 000个/孔,用于SU-DHL-2为28 000个/孔,最佳孵育时间为1h;alamarBlue可检测两种细胞的数目分别为14 000个/孔和16 000个/孔,最佳孵育时间分别为6h和2h;CellTiter-Glo可检测的细胞数目均为50 000个/孔,孵育时间为10min。结论 CCK-8和alamarBlue成本较低,孵育时间较长,线性范围较窄,实验前需要耗费时间摸索最佳条件;CellTiter-Glo线性范围较宽,检测效率和准确性最高,但是需要专门的检测设备,成本较高。
Objective To provide a comprehensive comparison and optimized protocol of three different cell activity detection reagents for two kinds of tumor cell. Methods The optimal conditions for the three common reagents of CCK-8, alamarBlue and CellTiter-Glo were explored respectively with adherent cell Hela and suspension cell SUDHL-2 as research subjects. Results For both kinds of cells, the three reagents had a good linear range under certain conditions, and all the Pearson correlation coefficient r>0.99. For CCK-8, the optimal alternative used to detect the Hela and SU-DHL-2 cell number were 8 000 cells per well and 28 000 cells per well respectively, and the best incubation time was 1 h. For alamarBlue, the optimal alternative used to detect the number of two kinds of cells were14 000 cells per well and 16 000 cells per well, and the optimal incubation time were 6 h and 2 h respectively. For CellTiter-Glo, up to 50 000 cells per well and 10 minutes incubation time had been detected. Conclusion CCK-8 and alamarBlue have lower cost, longer incubation time and narrower linear range, but the optimized protocol should be taken by pre-experiment with long time spending; CellTiter-Glo has a wide linear range, and the highest detection efficiency and accuracy, but the cost is higher because it requires special detection equipment.
Objective To provide a comprehensive comparison and optimized protocol of three different cell activity detection reagents for two kinds of tumor cell. Methods The optimal conditions for the three common reagents of CCK-8, alamarBlue and CellTiter-Glo were explored respectively with adherent cell Hela and suspension cell SUDHL-2 as research subjects. Results For both kinds of cells, the three reagents had a good linear range under certain conditions, and all the Pearson correlation coefficient r>0.99. For CCK-8, the optimal alternative used to detect the Hela and SU-DHL-2 cell number were 8 000 cells per well and 28 000 cells per well respectively, and the best incubation time was 1 h. For alamarBlue, the optimal alternative used to detect the number of two kinds of cells were14 000 cells per well and 16 000 cells per well, and the optimal incubation time were 6 h and 2 h respectively. For CellTiter-Glo, up to 50 000 cells per well and 10 minutes incubation time had been detected. Conclusion CCK-8 and alamarBlue have lower cost, longer incubation time and narrower linear range, but the optimized protocol should be taken by pre-experiment with long time spending; CellTiter-Glo has a wide linear range, and the highest detection efficiency and accuracy, but the cost is higher because it requires special detection equipment.
引文
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14 Panic G,Flores D,Ingram-Sieber K,et al.Fluorescence/luminescence-based markers for the assessment of Schistosoma mansoni schistosomula drug assays[J].Parasit Vectors,2015,8:624
2 LaLonde A,Romero-Creel MF,Lapizco-Encinas BH.Assessment of cell viability after manipulation with insulator-based dielectrophoresis[J].Electrophoresis,2015,36(13):1479-1484
3高丕尧,龙军先,甘嘉亮,等.CCK-8法与AlamarBlue法对RKO细胞株增殖活力测试条件的对比分析[J].华西药学杂志,2015,30(1):38-41
4 Nowak E,Kammerer S,Küpper JH.ATP-based cell viability assay is superior to trypan blue exclusion and XTT assay in measuring cytotoxicity of anticancer drugs Taxol and Imatinib,and proteasome inhibitor MG-132 on human hepatoma cell line HepG2[J].Clin Hemorheol Microcirc,2018,69(1-2):327-336
5李磊,杨雨晗,王双,等.细胞活性检测方法之比较[J].生物学杂志,2011,28(1):87-90
6 Kumar P,Nagarajan A,Uchil PD.Analysis of Cell Viability by the alamarBlue Assay[J].Cold Spring Harb Protoc,2018,6:95489
7王园园,谭小燕,胡明华,等.CCK-8法检测小鼠淋巴细胞增殖的条件探讨[J].药物评价研究,2017,40(2):206-209
8 Lomakina GY,Modestova YA,Ugarova NN.Bioluminescence assay for cell viability[J].Biochemistry(Mosc),2015,80(6):701-713
9邵军丽,李林秋,戴娟秀,等.CCK-8试剂用于测定活细胞数量的细节研究[J].生物技术世界,2016,5:31-32
10 Bonnier F,Keating ME,Wróbel TP,et al.Cell viability assessment using the Alamarblue assay:a comparison of 2D and3D cell culture models[J].Toxicol In Vitro,2015,29(1):124-131
11 Tolliday N.High-throughput assessment of Mammalian cell viability by determination of adenosine triphosphate levels[J].Curr Protoc Chem Biol,2010,2(3):153-161
12 Single A,Beetham H,Telford BJ,et al.A Comparison of RealTime and Endpoint Cell Viability Assays for Improved Synthetic Lethal Drug Validation[J].J Biomol Screen,2015,20(10):1286-1293
13 Eilenberger C,Kratz SRA,Rothbauer M,et al.Optimized alamarBlue assay protocol for drug dose-response determination of 3D tumor spheroids[J].Methods X,2018,5:781-787
14 Panic G,Flores D,Ingram-Sieber K,et al.Fluorescence/luminescence-based markers for the assessment of Schistosoma mansoni schistosomula drug assays[J].Parasit Vectors,2015,8:624