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
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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 by the third-generation flow cytometry sorter FACS Aria Ⅲ so as to further improve the optimization of parameter settings and conditions for single-cell microplate sorting.Methods:Single-cell sorting of four different types of cell lines-Raji cells,A549,DT40,RAW 264.7cell line-was done on FACSAria Ⅲ flow cytometry sorter and 96-well microplate.70μm,100μm and130μm diameter nozzles were adopted respectively,and the sorted single cell rate and the cell colony-forming rate were counted.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 μm,100μm and 130 μm 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 and optimization of parameters,100μm or 130μm nozzles should be given preference for single cell activity.
Objective:To compare the sorted single cell rate and cell viability with different diameters of nozzles by the third-generation flow cytometry sorter FACS Aria Ⅲ so as to further improve the optimization of parameter settings and conditions for single-cell microplate sorting.Methods:Single-cell sorting of four different types of cell lines-Raji cells,A549,DT40,RAW 264.7cell line-was done on FACSAria Ⅲ flow cytometry sorter and 96-well microplate.70μm,100μm and130μm diameter nozzles were adopted respectively,and the sorted single cell rate and the cell colony-forming rate were counted.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 μm,100μm and 130 μm 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 and optimization of parameters,100μm or 130μm nozzles should be given preference for single cell activity.
引文
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[3]CZECHOWSKA K,JOHNSON D R,VAN DER MEER JR.Use of flow cytometric methods for single-cell analysis in environmental microbiology[J].Curr Opin Microbiol,2008,11(3):205-212.
[4]KATSURAGI T,TANI Y.Screening for microorganisms with specific characteristics by flow cytometry and single-cell sorting[J].J Biosci Bioeng,2000,89(3):217-222.
[5]WIACEK C,MLLER S,BENNDORF D.A cytomic approach reveals population heterogeneity of Cupriavidus necator in response to harmful phenol concentrations[J].Proteomics,2006,6(22):5983-5994.
[6]MAGBANUA M J,PARK J W.Isolation of circulating tumor cells by immunomagnetic enrichment and fluorescenceactivated cell sorting(IE/FACS)for molecular profiling[J].Methods,2013,64(2):114-118.
[7]HAROON M F,SKENNERTON C T,STEEN J A,et al.In-solution fluorescence in situ hybridization and fluorescenceactivated cell sorting for single cell and population genome recovery[J].Methods Enzymol,2013,531:3-19.
[8]MORONO Y,TERADA T,KALLMEYER J,et al.An improved cell separation technique for marine subsurface sediments:applications for high-throughput analysis using flow cytometry and cell sorting[J].Environ Microbiol,2013,15(10):2841-2849.
[9]MANOME A,ZHANG H,TANI Y,et al.Application of gel microdroplet and flow cytometry techniques to selective enrichment of non-growing bacterial cells[J].FEMSMicrobiol Lett,2001,197(1):29-33.