FACSAria Ⅱ分选人肺腺癌A549细胞SP亚群的方法优化

Optimization of the method for sorting human lung cancer cell A549 with FACSAria Ⅱ

目的·通过优化流式分选人肺癌A549细胞SP亚群方法,提高SP亚群分选纯度并缩短分选时间,以提高分选活率。方法·制备单细胞悬液,不同终浓度维拉帕米孵育及Hoechst33342标记染色,经PI标记检测SP细胞比例。选择最佳染色浓度,100μm喷嘴,分选速度10μL/min,用一次分选法（纯度模式）和二次分选法（富集＋纯度模式）分选SP细胞和MP细胞,比较分选后纯度和分选用时。通过二次分选法获得SP细胞和MP细胞,分别行Lyso-Tracker Red/Hoechst 33342染色共聚焦观察和成瘤实验。结果·经二次分选法,SP细胞纯度达到99%以上,1×10～8个细胞分选时间〈5.2 h。共聚焦镜下观察到SP细胞大小与MP细胞无明显差异,MP细胞的溶酶体数量较SP细胞显著增多。Hoechst 33342均能染色,细胞活率不受影响。在小鼠成瘤实验中,SP细胞成瘤体积较MP细胞显著增大。结论·A549肺癌细胞Hoechst33342染色终质量浓度宜用6μg/mL;采用FACSAriaⅡ对其SP细胞进行二次分选,不仅显著提高SP细胞分选纯度,还大大缩短分选时间,细胞活率不受影响。

Objective · To improve the purity and survival rate and to reduce the time for sorting SP subpopulation of human lung cancer cell A549 by optimizing flow- cytometry sorting method. Methods · Single cell suspension was prepared, incubated with different concentration of Verapamil, and stained with different concentrations of Hoechst33342. The ratio of SP cells was detected following PI marking. One time sorting method （purity mode） and two time sorting method （enrichment and purity mode） were used to sort SP and MP cells with the best staining concentration, 100 μm nozzle, and sorting velocity of 10 μL/min. The sorting purity and time were compared between two methods. SP cells and MP cells sorted with the two time sorting method underwent tumor forming experiment and confocal observation after Lyso-Tracker Red and Hoechst 33342 stainings. Results · The purity of SP cells sorted with two times sorting method was more than 99% and the sorting time was less than 5.2 h for sorting 1×108 cells. Under confocal microscopy, the size of SP cells was not different from that of MP cells and MP cells had more lysosomal granules than SP cells. Both cells were able to be stained with Hoechst 33342 and the cell survival rate was not affected. In mice tumor forming experiment, the size of tumor formed with SP cells was significantly larger than that formed with MP cells. Conclusion · The optimal staining concentration of Hoechst33342 for A549 lung cancer cells is 6 μg/mL. Using two time sorting method and FACSAria Ⅱ flow sorter to sort SP cells can not only significantly improve the purity of SP cells, but also greatly reduce the sorting time without affecting the survival rate of SP cells.