CCK-8和MTS法检测人羊膜上皮细胞增殖的比较

Comparison of Experimental Conditions of CCK-8 and MTS for Human Amniotic Epithelial Cells Proliferation Assay

目的探讨CCK-8、MTS两种不同的四唑盐试剂在羊膜上皮细胞增殖检测中的最适实验条件,并比较两者细胞毒性。方法取体外培养对数生长期羊膜上皮细胞,用完全培养基（DMEM/F12＋10%胎牛血清）配制成不同浓度的细胞悬液加入96孔板中培养24 h,分别加入CCK-8、MTS后于450 nm、492 nm波长处测定光密度（OD）。在同一细胞浓度下,根据同一波长不同时间检测的OD确定CCK-8的最佳孵育时间。取体外培养的对数生长期羊膜上皮细胞分别用DMSO、CCK-8、MTS处理1 h、2 h、3 h和4 h后,继续培养24 h,在450 nm处以CCK-8检测细胞增殖,并通过台盼蓝染色计数活细胞数。结果 CCK-8法的最佳波长为450 nm,MTS法的最佳波长为492 nm。CCK-8法灵敏度稍低于MTS法。1~4 h内,CCK-8试剂与待检测细胞最佳孵育时间为4 h。CCK-8法对细胞增殖影响及细胞毒性均小于MTS法。结论 CCK-8法是一种更为方便、细胞毒性作用较小的试剂。

Objective To explore the optimal experiment conditions of CCK-8 and MTS for cell proliferation assays in human amniotic epithelial cells and to evaluate the cytotoxicity of these reagents.Methods Human amniotic epithelial cells（hAECs） in logarithm growth stages were prepared in different cell concentrations with DMEM/F12 and 10% FBS.The sensitivity and optimal wavelengths was determined based on the optical density（OD） measured at 450 nm and 492 nm.The optimal time was determined under the conditions of the same cell concentration and defined OD values.HAECs were treated with DMSO,CCK-8 and MTS for 1 h,2 h,3 h,and 4 h,respectively.24 h later,cytotoxicity of the CCK-8 and MTS was evaluated by determination of cell proliferation and Trypan Blue staining.Results The optimal detection wavelength was 450 nm for CCK-8,and 492 nm for MTS.The sensitivity of CCK-8 was slightly lower then that of MTS.The optimal time for incubation hAECs with CCK-8 was 4 h within 1~4 h.The inhibitory on cell proliferation and cytotoxicity of CCK-8 were weaker then those of MTS.Conclusion CCK-8 is a convenient reagent with low cytotoxicity for detection of the proliferation of hAECs.