流式细胞术分析苜蓿相对DNA含量的样品处理方法

为制备用于流式细胞仪分析的高纯度苜蓿(Medicago sativa)细胞核悬液,筛选最适合流式细胞仪分析的苜蓿细胞核提取方法,选用江苏地方品种淮阴苜蓿为供试材料,分别采用酶解法和机械解离法提取苜蓿细胞核悬液;用5种细胞核分离缓冲液及两种混合酶解液制备样品,根据各自优缺点筛选最适宜的苜蓿细胞核提取法。在机械法所用的5种细胞核提取缓冲液中,LB01缓冲液提取效果最好,提取的细胞核形态及内部结构完整,获得的细胞核量多。利用两种混合酶液制备苜蓿细胞核悬液,均出现倍性明显的峰,混合酶液Ⅰ制备的单细胞核悬液优于混合酶液Ⅱ,且酶解法制备细胞核悬液,可获得数量更多更完整的原生质体。但利用酶解法制备细胞核原生质体,其悬浮液中杂质多,需时较长。结果表明,LB01缓冲液为最适合制备苜蓿细胞核悬液的提取缓冲液。

Sample preparation for alfalfa relative DNA content analysis by flow cytometry

In order to select optimum extract methods for high purity alfalfa nuclear suspensions for flow cytometry(FCM), the nuclei suspension of Huaiyin alfalfa (Medicago sativa cv. huaiyin) was isolated either mechanically or by mixed enzyme solutions. The appropriate preparation method was selected based on the suspensions quality. LB01 buffer can extract larger number of nuclei with intact cell morphology and internal structure which performed best among these 5 buffers. Both of the nuclear suspensions prepared by two mixed enzyme solutions showed clear peaks on the histograms and had more nucleus. The nuclear suspension prepared by enzyme solution I was better than enzyme solution Ⅱ. However, the enzymatic methods were time-consuming and produced more impurities in the nuclear suspension. In conclusion, the mechanically extraction with LB01 buffer was appropriate method to prepare nulear suspensions for alfalfa nuclear DNA content analysis by FCM.