摘要
目的:通过分选肺腺癌A549细胞的侧群细胞(side population cells,SP),了解影响Influx分选SP的各种因素,为成功分选其他肿瘤细胞的SP亚群提供方法参考。方法:(1)细胞的制备:取对数生长期的细胞,制备单细胞悬液,用Hoechst33342及PI标记染色,同时设计维拉帕米对照组;(2)最佳分选喷嘴的确定:根据对数生长期的A549细胞处于不同的分裂时象的细胞大小,预实验确定最佳分选喷嘴;(3)光信号的调试:用Rainbow Beads优化670/30[355]、460/50[355]荧光信号和FSC散射光信号,使光信号最强且变异系数(coefficient of variation,CV)最小。(4)最佳液滴延迟的确定:调节振荡频率(frequency)找出最佳液流断点位置,调节振幅(ampitude)优化侧液流信号直到两侧液流信号最稳定,Delay Calculator自动计算液滴延迟,根据Accudrop Beads的分选效果确定最佳液滴延迟;(5)其他指标的调整:调节液滴的充电电压(drop charge)和分选装置的X、Y轴坐标,使测试分选液滴准确达到分选装置中;设置分选参数,分选目的细胞群。结果:同一批次处理的A549肺腺癌细胞,100μm的喷嘴为分选A549肺腺癌SP亚群的最佳喷嘴;CV值校正后SP比例为19.7%,提高了6%;液滴延迟校正后,其分选效率(efficiency)提高了15%,分选纯度(purity)提高了23%;10μg/mL的Hoechst33342染色,其SP比例是5μg/mL染色的5倍以上;细胞浓度越稀,分选效率和分选纯度相应提高;活细胞比例越高,SP比例越高。结论:合适的喷嘴大小,是保证高分选效率和高分选纯度的基础;仪器较小的CV值和较精确的液滴延迟、合理的Hoechst33342染料浓度、较高的细胞存活率和适中的细胞浓度是保证高分选效率和高分选纯度的的关键;通过平衡上述影响分选的因素才可能得到最佳的分选效果。
Objective：For successful separation of side population SP） cells of tumor cells by using BD Influx sorting instrument,the various factors considered were to be analyzed with using lung adenocarcinoma A549 cells as samples.Method：（1） Instrument conditions optimization：The most appropriate nozzle was chose according to the cell size （nozzle size =4 ～ 6 cell size）.The intensity and the coefficient of variation （CV） about excited fluorescence from rainbow beads at 670/30[355] and 460/50[355] were adjusted.After the drop frequency and the piezo amplitude were adjusted to get the optimal break off point and optimize the side streams,the drop delay was roughly estimated by delay calculator and the precise drop delay was determined by accudrop beads correction.The deflection charge amplitude and the X/Y-offsets were verified that the side streams to the center of tube.（2） Samples preparation：The cells in exponential phase were prepared to be as cell suspension cultured with Hoechst33342 and propidium iodide （PI）.Result：100 μm nozzle tip was the most appropriate nozzle tip for sorting SP population in A549 cells.CV decreased whereas the sorting purity and efficiency increased.The higher sorting purity and efficiency were associated with the more accurate drop delay and the smaller cell concentration.Further studies showed that the rate of SP population in tumor cells is related to the percentage of living cells positively.Conclusion：To assure sorting effective yield by using BD influx cell sorter,the mentioned factors must be considered synthetically.
出处
《激光生物学报》
CAS
CSCD
2013年第5期423-431,共9页
Acta Laser Biology Sinica
基金
广州市科技攻关项目(2010J-E261)
关键词
流式分选
侧群干细胞
变异系数
效率
纯度
FACS
sorting
side population cells
efficiency
purity
coefficient of variation
作者简介
周玉英（1981-）,女,汉族,山东鄄城县人,硕士,细胞生物学.（电话）020-85222759.
通讯作者：陈小佳（1973-）,女,汉族,广东潮州人,助理研究员,博士,硕士导师,细胞与分子生物学.（电话）020-85221983.
