快速诱导细胞凋亡方法探讨

The methods of inducing apoptosis rapidly

[目的]利用流式细胞仪检测细胞凋亡在基础研究、疾病诊断及预后预测及评价中具有重要的应用价值.补偿调节所需的单阳染色管的制备,是流式细胞术检测凋亡过程的一个重要步骤.探讨了快速诱导不同细胞系和原代细胞凋亡的方法,优化用于检测细胞凋亡的流式单阳染色管快速制备.[方法](1)分别用100μmol/L H_(2)O_(2)、体积分数4%多聚甲醛、75%乙醇和反复冻融的方法处理293T细胞15 min、30 min、1 h,Annexin V-PE/7-AAD双标记流式检测293T细胞凋亡率.(2)体积分数1%乙醇、5%乙醇、10%乙醇和20%乙醇处理293T细胞、RAW264.7细胞、HeLa细胞、T细胞和B细胞1 min,流式细胞仪检测细胞凋亡情况.[结果](1)293T细胞经100μmol/L H_(2)O_(2)、体积分数4%多聚甲醛、75%乙醇和反复冻融法处理后,几乎所有细胞均处于凋亡状态.(2)293T细胞、RAW264.7细胞、T细胞和B细胞用1%乙醇处理1 min,HeLa细胞用20%乙醇处理1 min,可出现较明显的凋亡现象.[结论]细胞凋亡实验时,根据细胞的生长特性,可选择体积分数1%~20%的乙醇诱导1 min,作为制备单阳染色管的最佳方案.

[Objective]Flow cytometry has the important application value to basic research,disease diagnosis,prognosis prediction and evaluation.The preparation of single positive staining tubes for compensation is a crucial step in the detection of apoptosis by flow cytometry.In this paper,the method of fast inducing apoptosis of different cell lines and primary cells was discussed.[Methods](1)293T cells were treated with 100μmol/L H_(2)O_(2),4%paraformaldehyde,75%ethanol,and freezing/thawing for 15 minutes,30 minutes and 1hour,respectively.Annexin V-PE/7-AAD dual-label was used to detect the rate of apoptosis by flow cytometry.(2)293T cells,RAW264.7 cells,HeLa cells,T cells and B cells were treated with 1%ethanol,5%ethanol,10%ethanol and 20%ethanol for one minute,and the apoptosis was detected by flow cytometry.[Results](1)After treated with 100μmol/L H_(2)O_(2),4%Polyformaldehyde,75%ethanol and freezing/thawing,293T cells were all apoptotic almostly.(2)293T cells,RAW264.7 cells,T cells,B cells were treated with 1%ethanol for one minute,HeLa cells were treated with 20%ethanol for one minute,which resulted in significant apoptosis.[Conclusion]According to the growth characteristics of cells,1%-20%of ethanol can be used to induce apoptosis,which is the best method to prepare single positive staining tube for detecting apoptosis by flow cytometry.