CCK-8法在淋巴细胞增殖检测中最佳实验条件的筛选
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摘要
目的筛选CCK-8法在淋巴细胞增殖检测中的最佳实验条件。方法采用正交实验设计,对初始细胞浓度、培养时间、LPS浓度、显色时间这4个主要因素各水平对人外周血单个核细胞(PBMC)和小鼠脾细胞增殖的影响进行试验研究,对各实验组合测得的刺激指数进行方差分析。结果 CCK-8检测人PBMC增殖试验的最佳条件:初始细胞浓度为2.5×10~6/mL,培养时间为48 h,LPS浓度为1μg/mL,加入CCK-8后孵育4.5 h;检测小鼠脾细胞增殖试验的最佳条件:初始细胞浓度为5.0×10~6/mL,培养时间为48 h,LPS浓度为1μg/mL,加入CCK-8后孵育4.5 h。结论 CCK-8法便捷、灵敏、重复性好,可作为检测淋巴细胞增殖的稳定方法。本研究建立的CCK-8最佳实验条件可为免疫调节作用的药物体外筛选和免疫药理学作用的研究提供依据。
Objective To optimize the experimental conditions of CCK-8 in lymphocyte proliferation assays. Methods An orthogonal test was designed to investigate the influence of four major factors(cell density, culture period, concentration of LPS and duration of incubation with CCK-8) on cell proliferation of human PBMC and mouse splenocyte.ANOVA was carried out to analyze the stimulation indices of all experimental condition combinations. Results The optimal conditions for CCK-8 was as follows: for PBMC, cell density was 2.5×10~6/mL, culture period was 48 h, concentration of LPS was 1 μg/mL, and duration of incubation with CCK-8 was 4.5 h; and for splenocyte, cell density was 5.0×10~6/mL, culture period was 48 h, concentration of LPS was 1 μg/mL, and duration of incubation with CCK-8 was 4.5 h.Conclusion The optimized CCK-8 protocol is a sensitive, convenient and stable quantitative method to evaluate lymphocyte proliferation. This result can provide evidence in screening of immunomodulating drugs and investigation of their immunopharmacology.
Objective To optimize the experimental conditions of CCK-8 in lymphocyte proliferation assays. Methods An orthogonal test was designed to investigate the influence of four major factors(cell density, culture period, concentration of LPS and duration of incubation with CCK-8) on cell proliferation of human PBMC and mouse splenocyte.ANOVA was carried out to analyze the stimulation indices of all experimental condition combinations. Results The optimal conditions for CCK-8 was as follows: for PBMC, cell density was 2.5×10~6/mL, culture period was 48 h, concentration of LPS was 1 μg/mL, and duration of incubation with CCK-8 was 4.5 h; and for splenocyte, cell density was 5.0×10~6/mL, culture period was 48 h, concentration of LPS was 1 μg/mL, and duration of incubation with CCK-8 was 4.5 h.Conclusion The optimized CCK-8 protocol is a sensitive, convenient and stable quantitative method to evaluate lymphocyte proliferation. This result can provide evidence in screening of immunomodulating drugs and investigation of their immunopharmacology.
引文
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[17]邓媛,陶善东,张欣,等.依鲁替尼与达沙替尼对急性淋巴细胞白血病细胞增殖的抑制及其机制实验研究[J].中国实验血液学杂志,2017,25(1):72-79.
[18]彭洪薇,张晓洁,李菲.黄芪多糖联合多柔比星抗白血病细胞HL-60/A耐药的机制研究[J].中国实验血液学杂志,2017,25(3):743-748.
[19]Jiao G,He X,Li X,et al.Limitations of MTT and CCK-8assay for evaluation of graphene cytotoxicity[J].Rsc Advances,2015,5(66):53 240-53 244.
[20]Meyer JS,Koehm SL,Hughes JM,et al.Bromodeoxyuridine labeling for S-phase measurement in breast carcinoma[J].Cancer,2015,71(11):3531-3540.
[21]Lin P,Allison DC.Measurement of DNA content and of tritiated thymidine and bromodeoxyuridine incorporation by the same cells[J].J Histochem Cytochem,1993,41(9):1435.
[2]Tian Z,An N,Zhou B,et al.Cytotoxic diarylheptanoid induces cell cycle arrest and apoptosis via increasing ATF3and stabilizing p53 in SH-SY5Y cells[J].Cancer Chemother Pharmacol,2009,63(6):1131-1139.
[3]Cory AH,Owen TC,Barltrop JA,et al.Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture[J].Cancer Commun,1991,3(7):207-212.
[4]Berridge MV,Tan AS,Mc Coy KD,et al.The biochemical and cellular basis of cell proliferation assays that use terazolium salts[J].Biochemica,1996,4:14-19.
[5]Zhang LW,Bumer W,Monteiroriviere NA.Cellular uptake mechanisms and toxicity of quantum dots in dendritic cells[J].Nanomedicine,2011,6(5):777-791.
[6]Jiang F,Liu T,He Y,et al.Mi R-125b promotes proliferation and migration of typeⅡendometrial carcinoma cells through targeting TP53INP1 tumor suppressor in vitro and in vivo[J].BMC Cancer,2011,11(1):425.
[7]Kan W,Hu X,Du C,et al.Angiotensin-(1-7)suppresses the number and function of the circulating fibrocytes by upregulating endothelial nitric oxide synthase expression[J].Mol Cell Biochem,2012,365(1-2):19-27.
[8]Miyamoto T,Min W,Lillehoj HS.Lymphocyte Proliferation Response during Eimeria tenella Infection Assessed by a New,Reliable,Nonradioactive Colorimetric Assay[J].Avian Diseases,2002,46(1):10-16.
[9]梁宏伟,朱雯宇,冯波,等.MTT法检测大鼠外周血淋巴细胞增殖反应探讨与应用[J].中国农学通报,2016,32(26):21-26.
[10]郭晓8),吴文慧,刘静亚,等.MTT法检测钛酸钡涂层对小鼠成纤维细胞L929增值率的影响[J].实用口腔医学杂志,2016,32(5):615-619.
[11]项峰,任利萍,刘克勤,等.子宫颈癌肿瘤干细胞筛选及体外敏感药物试验的临床对比研究[J].河北医药,2014,36(2):285-287.
[12]刘玺章,王代友,陈婷.口腔鳞状细胞癌体外药敏试验的临床研究[J].现代诊断与治疗,2016,27(2):239-240.
[13]Goodwin CJ,Holt SJ,Downes S,et al.Microculture tetrazolium assays:a comparison between two new tetrazolium salts,XTT and MTS[J].J Immunol Methods,1995,179(1):95-103.
[14]张会鲜,何琪杨.CCK-8法检测药物影响肿瘤细胞增殖的优化研究[J].药学研究,2016,35(2):63-66.
[15]赵艳坤,邵伟,雒诚龙,等.无血清共培养条件下脐带间充质干细胞对奶牛乳腺上皮细胞的增殖作用[J].中国实验动物学报,2017,25(4):391-398.
[16]蔡文涛.MTT法和CCK-8法检测中药抗病毒活性成分细胞毒性的比较[J].湖北大学学报:自然科学版,2017,39(3):305-310.
[17]邓媛,陶善东,张欣,等.依鲁替尼与达沙替尼对急性淋巴细胞白血病细胞增殖的抑制及其机制实验研究[J].中国实验血液学杂志,2017,25(1):72-79.
[18]彭洪薇,张晓洁,李菲.黄芪多糖联合多柔比星抗白血病细胞HL-60/A耐药的机制研究[J].中国实验血液学杂志,2017,25(3):743-748.
[19]Jiao G,He X,Li X,et al.Limitations of MTT and CCK-8assay for evaluation of graphene cytotoxicity[J].Rsc Advances,2015,5(66):53 240-53 244.
[20]Meyer JS,Koehm SL,Hughes JM,et al.Bromodeoxyuridine labeling for S-phase measurement in breast carcinoma[J].Cancer,2015,71(11):3531-3540.
[21]Lin P,Allison DC.Measurement of DNA content and of tritiated thymidine and bromodeoxyuridine incorporation by the same cells[J].J Histochem Cytochem,1993,41(9):1435.