CCK-8与MTT法检测左归丸含药血清干预BMSCs增殖条件的比较

Comparative study on experiment conditions between CCK-8 and MTT assay on BMSCs proliferation intervened by serum containing Zuoguiwan

目的探讨CCK-8与MTT法在检测左归丸含药血清干预大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)增殖时所需的最佳实验条件,并比较两种方法的优缺点。方法用左归丸含药血清进行干预P3 BMSCs,孵育结束后,分别加入CCK-8与MTT试剂,检测两种方法的最佳波长、培养时间、灵敏度和增殖时间。结果CCK-8法与MTT法检测BMSCs的最佳波长分别为450 nm和550 nm,孵育时间分别为3 h和4 h,细胞接种密度分别为0.1×10^(4)/孔和0.25×10^(4)/孔。CCK-8法与MTT法在BMSCs接种密度分别高于0.25×10^(4)/孔和0.5×10^(4)/孔时,两种方法检测所得OD值均提早进入平台期,易出现假S型增长曲线。而当密度分别低于0.05×10^(4)/孔和0.1×10^(4)/孔时,细胞增加缓慢,难以形成S型增长曲线,不能反映出BMSCs的增殖特性。与CCK-8法相比较,在细胞密度低于0.1×10^(4)/孔或高于2×10^(4)/孔时,MTT法对BMSCs在翻倍时OD值增加的响应量无明显变化,差异无统计学意义。而细胞密度为0.25×10^(4)/孔至1×10^(4)/孔时,MTT法对BMSCs在翻倍时OD值增加的响应量明显增加,差异有统计学意义(P<0.05)。结论MTT法更适用于左归丸含药血清干预BMSCs增殖和分化的研究。

Objective To explore the optimal conditions of cell counting kit-8 assay(CCK-8)and MTT assay on the detection of the proliferation of bone marrow mesenchymal stem cells(BMSCs)intervened by serum containing Zuoguiwan(ZGW).The advantages and disadvantages of the two method were compared.Methods BMSCs were intervened by serum containing ZGW.The optimal wavelength,culture time,sensitivity,and cell proliferation of BMSCs were detected by using CCK-8 and MTT method,respectively.Results Optimal detection wavelengths of CCK-8 and MTT reagents were 450 nm and 550 nm,respectively.The best incubation time of CCK-8 and MTT assay were 3 h and 4 h,respectively.Cell inoculation density should be respectively controlled by 0.1×10^(4)/hole and 0.25×10^(4)/hole,respectively.When the inoculation density of BMSCs was higher than 0.25×10^(4)/well and 0.5×10^(4)/well,the OD values detected by CCK-8 assay and MTT assay entered the plateau stage earlier.It was easy to appear the false S-shaped growth curve.However,when the cell density was lower than 0.05×10^(4)/well and 0.1×10^(4)/well,it was difficult to form S-shaped growth curve because of slow growing,which could not reflect the proliferation characteristics of BMSCs.Compared with CCK-8 assay,there was no significant change in response to the increase of OD value of BMSCs in MTT assay,when BMSCs density was lower than 0.1×10^(4)/well or higher than 2×10^(4)/well.When BMSCs density was between 0.25×10^(4)/well and 1×10^(4)/well,the response of MTT assay to the elevation of OD value of BMSCs increased significantly(P<0.05).Conclusion MTT assay is more suitable for the study of serum containing ZGW on the proliferation and differentiation of BMSCs.