利用流式分选技术富集空载质粒转染特定巨噬细胞的方法及条件优化
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摘要
目的:建立BD FACSAria II流式细胞分选仪富集PX458空载质粒转染J774A.1细胞的方法并进行技术优化,确立用于筛选J774A.1细胞的最佳条件。方法:构建PX458空载质粒转染J774A.1细胞株,进行7-AAD染色。选择BD FACSAria II流式细胞分选仪的100 μm喷嘴以及initial、4-way purity、purity、yield、fine tune、single cell 6种分选模式分选J774A.1细胞的GFP阳性转染株,比较仪器各部分参数变化对分选效率的影响。结果:在仪器稳定的液流条件下,100 μm喷嘴各分选模式所得细胞纯度均达80%以上,其中purity模式分选效率最高,达95.7%±1.0%。结论:使用BD FACSAria II流式细胞分选仪100 μm喷嘴分选PX458空载转染的J774A.1细胞选择purity分选模式最合适。
Objective: To establish and optimize the method for enriching PX458 empty plasmid transfected J774 A.1 cells by BD FACSAria II flow cytometer in order to figure out the optimum condition for screening J774 A.1 cells. Methods: PX458 empty plasmid transfected J774 A.1 cell line was constructed and stained with 7-AAD. GFP-positive transfectants of J774 A.1 cells were sorted by 100 μm nozzle and initial, 4-way purity,purity, yield, fine tune, single cell sort modes respectively. The sorting efficiency was compared in different instrument parameters. Results: Under the stable fluid flow condition, the purity of the cells obtained by each sort mode of 100 μm nozzle was over 80%, and the sort mode with highest efficiency is purity mode, reaching95.7%±1.0%. Conclusion: When the PX458 empty plasmid transfected J774 A.1 cell line was shorted by BD FACSAria II flow cytometer with 100 μm nozzle, the purity sort mode is preferred.
Objective: To establish and optimize the method for enriching PX458 empty plasmid transfected J774 A.1 cells by BD FACSAria II flow cytometer in order to figure out the optimum condition for screening J774 A.1 cells. Methods: PX458 empty plasmid transfected J774 A.1 cell line was constructed and stained with 7-AAD. GFP-positive transfectants of J774 A.1 cells were sorted by 100 μm nozzle and initial, 4-way purity,purity, yield, fine tune, single cell sort modes respectively. The sorting efficiency was compared in different instrument parameters. Results: Under the stable fluid flow condition, the purity of the cells obtained by each sort mode of 100 μm nozzle was over 80%, and the sort mode with highest efficiency is purity mode, reaching95.7%±1.0%. Conclusion: When the PX458 empty plasmid transfected J774 A.1 cell line was shorted by BD FACSAria II flow cytometer with 100 μm nozzle, the purity sort mode is preferred.
引文
[1]周密,张全胜,包明柱.流式细胞仪细胞分选参数调校方法的研究[J].医疗装备,2016,29(7):58-61.
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[11]张晖,周春保.FACS Aria流式细胞分选仪质量控制体系的优化[J].中国医疗器械杂志,2009,33(3):225-227.
[12]刘锡娟,丁慧荣,田志华,等.FACSAria流式细胞仪无菌操作分选高纯度细胞亚群[J].安徽医科大学学报,2014,49(12):1811-1815.
[13]SABATH D E,SHIM M H.Use of green fluorescent protein/Flp recombinase fusion protein and flow cytometric sorting to enrich for cells undergoing Flp-mediated recombination[J].Biotechniques,2000,28(5):966-972,974.
[2]杨红艳,焉力方,王如刚,等.应用流式细胞技术分选单核细胞亚群不同预富集方法的探讨[J].现代检验医学杂志,2018,33(1):128-130.
[3]高一龙,秦海斌,温海,等.拉布拉多大X/Y精子分选及性别控制冷冻精液制备实验研究[J].实验动物与比较医学,2015,35(6):484-489.
[4]SHIBER A,BREUER W,RAVID T.Flow cytometric quantification and characterization of intracellular protein aggregates in yeast[J].Prion,2014,8(3):276-284.
[5]郭陈智,史桂英.流式分选仪FACSAria II分选GFP阳性乳腺癌MCF-7细胞株的条件优化研究[J].上海交通大学学报(医学版),2013,33(1):127-130.
[6]闵智慧,程韵枫.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[J].中国临床医学,2016,23(6):846-850.
[7]黄莹莹,尹盛夏,曹文强,等.高端流式分选与免疫磁珠法纯化T细胞的比较[J].国际医药卫生导报,2016,22(22):3377-3383.
[8]VOROBJEV I A,BUCHHOLZ K,PRABHAT P,et al.Optimization of flow cytometric detection and cell sorting of transgenic plasmodium parasites using interchangeable optical filters[J].Malar J,2012,11(1):1-10.
[9]曹云新,胡金涛,杨安钢.FACS-Aria流式细胞分选调试参数和最佳条件的设定[J].医学争鸣,2007,28(19):1813-1815.
[10]TIROUVANZIAM R,DAVIDSON C J,LIPSICK J S,et al.Fluorescence-activated cell sorting(FACS)of drosophila hemocytes reveals important functional similarities to mammalian leukocytes[J].Proc Natl Acad Sci U S A,2004,101(9):2912-2917.
[11]张晖,周春保.FACS Aria流式细胞分选仪质量控制体系的优化[J].中国医疗器械杂志,2009,33(3):225-227.
[12]刘锡娟,丁慧荣,田志华,等.FACSAria流式细胞仪无菌操作分选高纯度细胞亚群[J].安徽医科大学学报,2014,49(12):1811-1815.
[13]SABATH D E,SHIM M H.Use of green fluorescent protein/Flp recombinase fusion protein and flow cytometric sorting to enrich for cells undergoing Flp-mediated recombination[J].Biotechniques,2000,28(5):966-972,974.