Studies of cell division (mitosis and cytokinesis) by dynamic secondary ion mass spectrometry ion microscopy:: LLC-PK1 epithelial cells as a model for subcellular isotopic imaging
Studies of cell division (mitosis and cytokinesis) by dynamic secondary ion mass spectrometry ion microscopy:: LLC-PK1 epithelial cells as a model for subcellular isotopic imaging
复制标题
DOI：
10.1046/j.1365-2818.2001.00944.x
复制
发表时间：
2001-11-01
期刊：
影响因子：
--
通讯作者：
Chandra, S
中科院分区：
文献类型：
--
作者：
Chandra, S
用二次离子质谱仪(SIMS)验证了肾上皮细胞系LLC-PK1作为细胞分裂研究模型的可行性。在该细胞系中，经历有丝分裂和胞质分裂的所有阶段的细胞仍然牢固地附着在底物上，并可以进行低温准备。断裂的冻干有丝分裂细胞显示细胞器保存完好，激光共聚焦扫描显微镜显示罗丹明-123和C-6-NBD-神经酰胺的荧光成像。断裂的冻干分裂细胞的二次电子显微镜分析显示最小的表面形貌，不干扰两种阳性(K-39，Na-23，Mg-24，Ca-40等)的同位素成像。阴性(P-31、Cl-35等)用Cameca IMS-3f离子显微镜观察。有丝分裂细胞显示，即使是最易扩散的离子(K-39(-/-)和Na-23(+)的总浓度)也保存完好，K-39(-/-)和Na-23(+)的总浓度约为10。有丝分裂细胞的结构损伤可以通过其降低的K：Na比率和过量的钙负载来识别。为了研究分裂细胞中亚细胞内钙的分布，需要进行定量的三维SIMS分析。LLC-PK1模型还允许SIMS研究使用抑制有丝分裂的药物(紫杉醇、莫那曲尔和诺可达唑)的M期停滞细胞。这项研究为利用SIMS研究细胞分裂的离子通量和化学组成开辟了新的途径。
The feasibility of the renal epithelial LLC-PK1 cell line as a model for cell division studies with secondary ion mass spectrometry (SIMS) was tested. In this cell line, cells undergoing all stages of mitosis and cytokinesis remained firmly attached to the substrate and could be cryogenically prepared. Fractured freeze-dried mitotic cells showed well-preserved organelles as revealed by fluorescence imaging of rhodamine-123 and C-6-NBD-ceramide by confocal laser scanning microscopy. Secondary electron microscopy analysis of fractured freeze-dried dividing cells revealed minimal surface topography that does not interfere in isotopic imaging of both positive (K-39, Na-23, Mg-24, Ca-40, etc.) and negative (P-31, Cl-35, etc.) secondaries with a CAMECA IMS-3f ion microscope. Mitotic cells revealed well-preserved intracellular ionic composition of even the most diffusible ions (total concentrations of K-39(-/-) and Na-23(+)) as revealed by K : Na ratios of approximately 10. Structurally damaged mitotic cells could be identified by their reduced K : Na ratios and an excessive loading of calcium. Quantitative three-dimensional SIMS analysis was required for studying subcellular calcium distribution in dividing cells. The LLC-PK1 model also allowed SIMS studies of M-phase arrested cells with mitosis-arresting drugs (taxol, monastrol and nocodazole). This study opens new avenues of cell division research related to ion fluxes and chemical composition with SIMS.