Department of Cytology and Histology, Institute of Zoology, Jagiellonian University, Krakow, Poland.
Fish Physiol Biochem. 2012 Apr;38(2):355-62. doi: 10.1007/s10695-011-9512-1. Epub 2011 May 26.
The aim of this study was to compare DNA content in hepatocyte and erythrocyte nuclei of the European sunbleak, Leucaspius delineatus, in relation to nuclear and cell size by means of flow cytometry and fluorescence microscopy. The DNA standards, chicken and rainbow trout erythrocytes, were prepared in parallel with both cell types, with initial separation of liver cells in pepsin solution followed by cell filtering. Standards and investigated cells were stained with a mixture of propidium iodide, citric acid, and Nonidet P40 in the presence of RNAse, and fluorescence of at least 50,000 nuclei was analyzed by flow cytometry. Average cell size was determined by flow cytometry, using fresh cell suspension in relation to latex beads of known diameter. The size of nuclei was examined on the basis of digital micrographs obtained by fluorescence microscopy after nuclei staining with DAPI. The sunbleak's erythrocyte nuclei contain 2.25 ± 0.06 pg of DNA, whereas the hepatocyte nuclei contain 2.46 ± 0.06 pg of DNA. This difference in DNA content was determined spectroscopically using isolated DNA from the two cell types. The modal diameters of the erythrocytes and hepatocytes were estimated to be 5.1 ± 0.2 and 22.3 ± 5.0 μm, respectively, and the corresponding modal dimensions of their nuclei (measured as surface area) were 15.2 and 21.4 μm(2), respectively. The nucleoplasmic index, as calculated from diameters estimated from surface area of nuclear profiles, was 2.51 for the erythrocytes compared with 0.08 for hepatocytes.
本研究旨在通过流式细胞术和荧光显微镜比较欧洲无须鳕（Leucaspius delineatus）肝细胞核和红细胞核中的 DNA 含量与核和细胞大小的关系。使用胃蛋白酶溶液分离肝细胞，然后进行细胞过滤，与两种细胞类型平行制备 DNA 标准品（鸡和虹鳟鱼的红细胞）。用碘化丙啶、柠檬酸和非离子型去垢剂混合物对标准品和被检测细胞进行染色，在 RNAse 存在的情况下，对至少 50,000 个核的荧光进行流式细胞术分析。使用新鲜细胞悬浮液与已知直径的乳胶珠的关系，通过流式细胞术确定平均细胞大小。用 DAPI 对细胞核进行染色后，通过荧光显微镜获得数字显微照片，基于这些照片来检查细胞核的大小。无须鳕的红细胞核含有 2.25 ± 0.06 pg 的 DNA，而肝细胞核含有 2.46 ± 0.06 pg 的 DNA。使用两种细胞类型的分离 DNA 通过光谱法确定了这种 DNA 含量的差异。红细胞和肝细胞的模态直径估计分别为 5.1 ± 0.2 μm 和 22.3 ± 5.0 μm，相应的核模态尺寸（以表面积测量）分别为 15.2 和 21.4 μm(2)。从核轮廓表面积估计的直径计算出的核质指数，红细胞为 2.51，而肝细胞为 0.08。
