CCK-8法检测小鼠淋巴细胞增殖的条件探讨

Study on determination conditions for lymphocytic proliferation by CCK-8 method in mice

目的探索CCK-8法检测小鼠脾淋巴细胞增殖的最佳条件。方法采用交叉设计或两因素完全随机设计探索分裂素刀豆蛋白(ConA)或脂多糖(LPS)刺激的脾细胞增殖实验的细胞制备方法、细胞浓度、分裂素浓度、刺激时间、培养基中胎牛血清(FBS)浓度、是否预培养等条件。结果制备方法中以轻压制备的脾细胞增殖率高于轻磨;脾细胞浓度以5×106/m L为最佳;ConA浓度为2、5、10μg/m L,LPS浓度为10、20、50μg/m L细胞增殖率未见明显差异;刺激时间在48和72 h间脾细胞增殖率未见明显差异;用含10%、15%和20%FBS的培养基培养的脾细胞增殖率未见明显差异;制备的脾细胞当天刺激比第二天刺激增殖率更高。结论 ConA和LPS刺激的Balb/C小鼠脾细胞增殖实验的最佳条件为,轻压制备脾细胞,脾细胞浓度为5×106/m L,制备当天直接种板,ConA的刺激浓度为2~10μg/m L,LPS的刺激浓度为10~50μg/m L。

Objective To study the optimum determination conditions for lymphocytic proliferation by CCK-8 method in mice. Methods To study the different influence factors of spleen cell proliferation experiment stimulated by mitogen concanavalin A（ConA） or lipopolysaccharide（LPS）, including cell preparation method, lymphocytic density, FBS and stimulating agent concentration in culture medium, and stimulating immediately or 24 h after preparing cell, with cross design or two factor completely randomized design. Results Spleen lymphocytic proliferation rate of preparation method by light suppression was higher than that of the light grind. The appropriate concentration of spleen cells was 5 × 10^6 /m L. The proliferation rate has no significant difference after being stimulated for 48 or 72 h by ConA（2, 5, or 10 μg/m L） or LPS（10, 20, or 50 μg/m L） under 10%, 15%, or 20% FBS concentration in culture medium. The proliferation rate of stimulating immediately after preparing cell was higher than that of 24 h after preparing cell. Conclusion The optimum conditions of Balb/C mouse spleen cell proliferation assay stimulated by ConA and LPS are as follows： preparation of spleen cells with light pressure, spleen cell concentration of 5×10^6/m L, direct stimulation with 2—10 μg/m L ConA or 10—50 μg/m L LPS in the day of preparation.