CCK-8法在淋巴细胞增殖检测中最佳实验条件的筛选

Optimization of the experimental conditions of CCK-8 in lymphocyte proliferation assays

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.