基于流式细胞仪分析的欧洲云杉原胚团细胞核提取液筛选

Nuclear suspension is an important foundation for a variety of molecular biology researches. Toeffectively prepare the nuclear suspensions of Norway spruce (Pice aabies) proembryogenic masses (PEM),PEMs of Norway spruce embryonic cell lines PaVIII, PI3, and PI5were used as the material for nuclearextraction with six lysis buffers: (LB01, Ottos, Galbraiths, GPB, WPB and TrisMgCl2). Based on detectingDNA content by flow cytometry, the quantity and quality of the nuclei suspensions were evaluated to screenfor the optimal buffer. Further, the integrity of nucleus and the stability of optimal buffer were measured. Theresults were as follows: (1) Compared with the other lysates, the number of nuclei obtained of PaVIII linePEMs from LB01 was the highest, but lower amount of cell debris. (2) In addition, coefficients of variation(CVs) of G0/G1 and G2/M nuclei, obtained from LB01, were not more than 5%. The CVs were relativelylow. (3) The nuclei of different lines PEMs were extracted by LB01. There was no significant difference inthe efficiency of nucleus extraction and the proportion of cell debris among the different lines. (4) Thenuclear membrane of most cells maintained in a high integrity when the nuclei of Norway spruce PEMswere extracted via LB01 through morphology investigation. To sum up, compared with other buffers, LB01is the optimal one for Norway spruce PEMs nuclear extraction, which might provide reference for nuclearextraction from gymnosperms.