DAPI染色法在流式细胞术中去除死细胞影响的探讨

目的:探索利用DAPI染色法在流式细胞检测过程中去除死细胞,消除死细胞对流式结果的影响。方法:建立原位肝癌小鼠模型,流式检测其中死细胞对脾脏中骨髓来源抑制性细胞(myeloid-derived suppressor cell,MDSC)结果的影响,并以碘化丙啶(propidiumiodide,PI)为对照,探索不同DAPI浓度、不同染色时间对死细胞比例的影响。结果:流式细胞术中去除死细胞影响时DAPI染色浓度低于1 g/m L时,其染色结果影响不大,浓度大于1 g/m L,死细胞比例明显升高;当DAPI使用浓度为0.2 g/m L时,在30钟内死细胞染色结果与PI相比差别无统计学差异,当染色时间大于30 min时,死细胞的比例显著升高。结论:DAPI染色法应用于流式细胞术中去除死细胞是一种可靠的检测方法,且简单易行,推荐使用方法为0.2 g/m L,染色时间1-5 min。

A Methodology Study on Elimination the Influence of Dead Cells Stained with DAPI

ABSTRACT Objective: To explore the method of DAPI staining in the process of flow cytometry to remove dead cells and elimi- nate the influence of dead cells on flow cytometry results. Methods: An orthotopic transplantion tumor model of hepatoma in mice was used to detect the dead cells effect on the result of MDSC in splencyte by flow cytometry. PI was used as a positive control to contrast the influence of different DAPI concentration and staining time on the proportion of dead cells. Results: The data showed that while the DAPI concentration was less than 1 g/mL, there had a similar result of the proportion of dead cells compared with PI staining; however, if the concentration of DAPI was more than 1 g/mL, the proportion of dead cells was increased. Stained the cells with 0.2 g/mL of DAPI within 30 minutes had a little influence on the proportion of dead cells. But when the staining time exceed 30 minutes, the proportion of dead cells increased obviously. Conclusion: The DAPI staining method is a reliable and easy detection method applied to remove dead cells effect on the flow cytometry, 0.2 g/mL of DAPI stained 1-5 minutes was recommend.