摘要
流式细胞分选是纯化活细胞较为常用的实验方法,是获得高活性细胞的有效手段,其中收集液为分选后的细胞提供一定的营养环境,从而保证分选后细胞的高活性。该研究选用流式细胞分选仪分选人肾上皮细胞系HEK293T(简称293T)细胞,用3组即A组(含BSA的PBS缓冲液)、B组(含FBS的PBS缓冲液)、C组(含FBS的DMEM培养基)收集液分别接收分选后的293T细胞,以流式细胞检测法、细胞计数试剂盒(Cell Counting Kit-8,CCK-8)检测法、实时细胞分析(Real Time Cellular Analysis,RTCA)检测法分别检测分选后细胞的活性情况并进行统计分析。结果显示,A组中含0.4% BSA的PBS缓冲液、B组中含10% FBS的PBS缓冲液、C组中含10% FBS的DMEM培养基接收的细胞活性分别高于各组内的对照,凋亡率分别为(4.12±0.474)%、(4.10±0.660)%、(3.71±0.291)%,均具有显著性统计学差异(P<0.000 1)。比较三种细胞活性检测方法发现,RTCA检测法具有极简的操作流程,无须标记,可实时获得细胞的动态增殖曲线的优点,可作为分选后细胞活性最客观的检测方法。该研究为揭示收集液对细胞活性的影响提供参考依据,为快速客观检测分选后细胞活性提供实时检测方法。
Flow cytometry sorting is a commonly used experimental method for purification of living cells and an effective means to obtain highly active cells.The collection solution provides a certain nutrient environment for the sorted cells,thus ensuring the high activity of the sorted cells.This study used flow cytometer to sort human renal tubular epithelial 293T (HEK293T) cells and received them with three groups of collection solution:group A (BSA in PBS buffer),group B (FBS in PBS buffer) and group C (FBS in DMEM medium).Flow cytometry,CCK-8 (Cell Counting Kit-8),and RTCA (Real Time Cellular Analysis) detection methods were used to detect the activity of the sorted cells,and then statistical analysis was performed.The results showed that the cell viabilities of group A (0.4% BSA in PBS buffer),group B (10% FBS in PBS buffer) and group C (10% FBS in DMEM medium) are higher than those of control in each group.The apoptosis rates separately were (4.12±0.474)%,(4.10±0.660)%,(3.71±0.291)%,with significant statistical differences (P<0.000 1).Comparison of the three cell viability detection methods showed that the RTCA detection method had the advantages of a minimalist operation process,no labeling,and real-time acquisition of the dynamic proliferation curve of cells,which could be used as the most objective detection method for cell viability after sorting.This study provides a reference for revealing the influence of the collection solution on cell viability,and a real-time detection method for rapid and objective detection of cell viability after sorting.
作者
李艳伟
宋兴辉
邢月婷
王佳佳
郭春
黄莹莹
LI Yanwei;SONG Xinghui;XING Yueting;WANG Jiajia;GUO Chun;HUANG Yingying(Core Facilities,School of Medicine Zhejiang University,Hangzhou 310058,China)
出处
《中国细胞生物学学报》
CAS
CSCD
2021年第10期2009-2020,共12页
Chinese Journal of Cell Biology
基金
浙江省教育厅基金(批准号:Y201941524)资助的课题。
关键词
CCK-8
流式细胞分析
实时检测
流式细胞分选
细胞活性
收集液
CCK-8
flow cytometric analysis
real time detection
flow cytometric sorting
cell viability
collection solution
