Calcein-AM荧光扫描测定细胞毒方法的建立  

Development of a novel assay for cytotoxicity test by Calcein-AM release

作　　者：陶嫦立 丁哲纯 郭雯静[1] 吴凤麟[1] 邵红伟[1] 黄树林[1] 

机构地区：[1]广东药学院生命科学与生物制药学院/广东省生物技术候选药物研究重点实验室,广州510006

出　　处：《中国免疫学杂志》2014年第10期1364-1368,共5页Chinese Journal of Immunology

基　　金：国家自然科学基金(31100664);国家重大科技专项"重大新药发展"(2009ZX09103-708)项目资助

摘　　要：目的：建立和优化基于钙黄绿素-AM（Calcein-AM）荧光扫描的细胞毒测定方法。方法：首先使用Calcein-AM染色靶细胞,扫描检测Calcein-AM荧光值,确定Calcein-AM最佳激发波长和发射波长;然后优化Calcein-AM染色的浓度和时间;最后以效靶比为30∶1～1∶1,共孵育时间为4 h,荧光扫描测定靶细胞裂解的百分率。结果：荧光染料Calcein-AM能有效标记靶细胞,最佳激发波长和发射波长为485 nm和515 nm;Calcein-AM对细胞毒性低,不影响细胞活性;4 h内的自发释放率低于15%;CIK（Cytokine induced killer）效应细胞与靶细胞共孵育4 h的细胞毒效应随效靶比增加而升高。结论：建立和优化了Calcein-AM荧光染料标记靶细胞检测细胞毒效应的方法,该方法可避免放射性试剂的应用,对细胞毒性低,准确性高。Objective： To develop and optimize a novel assay for determination of cytotoxicity based Calcein-AM release. Methods： The target cells stained by Calcein-AM dye,then effectors and targets were incubated at E /T ratios from 30∶ 1- 1∶ 1for 4 h at 37℃,and the supernatant of reactions were detected by Fluorescence-Measurement to analyze specific cytotoxity. Results：The optimal excitation and emission wave lengths of Calcein were 485 nm and 515 nm. Dilutions of target cells stained by Calcein-AM had a linear relationship with measured fluorescence values. The Calcein-AM dye used to stain the living cells was shown to have a low spontaneous leakage rate-less than 15% in 4 hours at 37℃. Cytotoxicity activity of CIK showed a significant and positive correlation with E /T ratio when incubated at 4 h. Conclusion： The developed cytotoxicity test by Calcein-AM release is accurate and can avoid the application of radioactive reagents.

关 键 词：Calcein-AM 细胞毒 荧光释放测定 

分 类 号：R392.33[医药卫生—免疫学]