第三代流式细胞分选仪及96孔板分选单个细胞的方法及参数优化  

Method and parameter optimization of the third-generation flow cytometry sorter and 96-well plate in single cell sorting

作　　者：闵智慧[1,2,3] 程韵枫[1,4,2,5,3] MIN Zhi-hui CHENG Yun-feng(Experimental Research Center of Institute for Clinical Sciences, Zhongshan Hospital, Fudan University, Shanghai200032, China Department of Hematology, Zhongshan Hospital, Fudan University, Shanghai 200032, China Experimental Research Center, Qingpu Branch of Zhongshan Hospital, Fudan University, Shanghai 201700, China Department of Hematology, Qingpu Branch of Zhongshan Hospital, Fudan University, Shanghai 201700, China Shanghai Key Laboratory of Organ Transplantation, Shanghai 200032, China)

机构地区：[1]复旦大学附属中山医院临床研究院实验研究中心,上海200032 [2]复旦大学附属中山医院青浦分院实验研究中心,上海201700 [3]上海市器官移植重点实验室,上海200032 [4]复旦大学附属中山医院血液科,上海200032 [5]复旦大学附属中山医院青浦分院血液科,上海201700

出　　处：《中国临床医学》2016年第6期846-850,共5页Chinese Journal of Clinical Medicine

基　　金：国家自然科学基金(81170473)~~

摘　　要：目的：用第三代流式细胞分选仪比较不同直径喷嘴分选的单细胞的得率及细胞活性,进一步完善单细胞微孔板分选的参数设置及条件优化。方法：用FACSAriaⅢ流式细胞分选仪及96孔板对Raji细胞、A549、DT 40、RAW 264.7细胞系进行单细胞分选,分别采用70μm、100μm和130μm喷嘴分选,比较分选后单细胞得率及克隆形成率。结果：在单细胞分选模式下,用3种不同直径喷嘴及96孔板分选后的单细胞得率为86.55%-93.44%,即96孔板有单细胞的孔数为84-92孔;分选后的单个细胞培养7d后,经70μm、100μm和130μm喷嘴分选的单细胞克隆孔数分别为31-52孔、49-70孔、58-78孔。结论：进行单细胞微孔板分选时,在精确调节及优化实验参数的前提下,为了保证单细胞的活性,应优先选择100μm或130μm喷嘴。Objective ： To compare the sorted single cell rate and cell viability with different diameters of nozzles bythe third-generation flow cytometry sorter FACS Aria El so as to further improve the optimization of parameter settings andconditions for single-cell microplate sorting. Methods： Single-cell sorting of four different types of cell lines-Raji cells, A549,D T40, RAW 264. 7 cell line-was done on FACSAria El flow cytometry sorter and 96-well microplate. 70 pm, 100 pm and130 jitm diameter nozzles were adopted respectively, and the sorted single cell rate and the cell colony-forming rate werecounted. Results： In the single-cell sorting mode, the percentages of sorted single cell with three nozzles were from 86. 55% to93. 44% , which meant that the hole numbers with single cell in the 96-well micorplate were from 84 to 92. After 7-day culture,Single cell clone hole numbers sorted by 70 pm, 100 pcm and 130 pm diameter nozzles were 31-52,49-70 and 58-78,respectively. Conclusions： In single cell sorting with the microplate and under the premise of accurate adjustment andoptimization of parameters, 100 jitm or 130 jitm nozzles should be given preference for single cell activity.

关 键 词：单细胞分选 流式细胞分选仪 96孔板 喷嘴 液滴延迟 

分 类 号：Q233[生物学—细胞生物学]