IMAGING INTRACELLULAR ELEMENTAL DISTRIBUTION AND ION FLUXES IN CULTURED-CELLS USING ION MICROSCOPY - A FREEZE-FRACTURE METHODOLOGY
IMAGING INTRACELLULAR ELEMENTAL DISTRIBUTION AND ION FLUXES IN CULTURED-CELLS USING ION MICROSCOPY - A FREEZE-FRACTURE METHODOLOGY
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DOI：
10.1111/j.1365-2818.1986.tb04670.x
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发表时间：
1986-10-01
影响因子：
2
通讯作者：
WOLCOTT, CC
中科院分区：
文献类型：
--
作者：
CHANDRA, S;MORRISON, GH;WOLCOTT, CC
一个冷冻断裂方法标准化的组织培养细胞，研究细胞内分布的扩散元素与离子显微镜。在存在间隔物的情况下，使用另一个光滑表面（硅、玻璃、云母）将在硅基质上生长的中国仓鼠卵巢（CHO）和正常大鼠肾（NRK）细胞夹在中间，并在液氮泥浆中快速冷冻。通过在液氮下将两半分开来使夹层破裂。该程序在硅基质上产生了包含数百个细胞的大面积，这些细胞聚集在一起并在顶端细胞表面破裂。冷冻干燥后，这些细胞显示Na、K、Ca、Mg、P、Cl和S的亚细胞分布，0.5离子显微镜的横向分辨率为μ m。在细胞核和细胞质之间，观察到Na、K、Mg、P、Cl和S的强度没有大的差异。然而，单独的钙表现出显著的分布。钙在细胞质中的积累多于细胞核。即使在细胞质内，其分布是异质性的，这表明钙结合位点。断裂的细胞始终表现出高K-低Na强度。损伤或死亡的细胞由于其异常的离子组成而容易在健康细胞中被识别。这种简单的冷冻断裂方法允许细胞断裂而不从基底移除细胞。此外，它消除了在离子微量分析之前冲洗营养培养基和冷冻切片的需要。该方法成功地扩展到3T3小鼠成纤维细胞，PtK2大鼠袋鼠和L5大鼠成肌细胞培养物。
A freeze-fracture methodology was standardized for tissue culture cells to study intracellular distribution of diffusible elements with ion microscopy. Chinese hamster ovary (CHO) and normal rat kidney (NRK) cells grown on a silicon substrate were sandwiched using another smooth surface (silicon, glass, mica) in the presence of spacers and fast frozen in liquid nitrogen slush. The sandwich was fractured by prying the two halves apart under liquid nitrogen. This procedure produced large areas on the silicon substrate containing hundreds of cells grouped together and fractured at the apical cell surface. After freeze-drying, these cells revealed a subcellular distribution of Na, K, Ca, Mg, P, Cl and S with the .apprx. 0.5 .mu.m lateral resolution of the ion microscope. Between the nuclei and the cytoplasm of cells, no major differences were observed for Na, K, Mg, P, Cl and S intensities. Calcium alone, however, exhibited a remarkable distribution. Calcium accumulated more in the cytoplasm than in the nuclei of cells. Even within the cytoplasm its distribution was heterogeneous, suggesting Ca binding sites. The fractured cells consistently exhibited high K-low Na intensities. The injured or dead cells were easily recognized among the healthy ones due to their abnormal ion composition. This simple freeze-fracture methodology allowed fracturing of cells without removing the cells from the substrate. In addition, it eliminated the need for washing the nutrient media away and cryo-sectioning before ion microanalysis. The methodology was successfully extended to 3T3 mouse fibroblast, PtK2 rat kangaroo and L5 rat myoblast cultures.