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首页> 外文期刊>Journal of Microscopy >A procedure to prepare cultured cells in suspension for electron probe X-ray microanalysis: application to scanning and transmission electron microscopy.
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A procedure to prepare cultured cells in suspension for electron probe X-ray microanalysis: application to scanning and transmission electron microscopy.

机译：一种制备悬浮细胞以进行电子探针X射线微分析的细胞的方法：在扫描和透射电子显微镜中的应用。

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摘要

We describe a simple procedure to prepare cultured cells in suspension to analyse elemental content at the cellular level by electron probe X-ray microanalysis. Cells cultured in suspension were deposited onto polycarbonate tissue, culture plate well inserts, centrifuged at low g, washed to remove the extracellular medium, cryofixed and freeze-dried, and analysed in the scanning mode of a scanning electron microscope. We tested the effect of different washing solutions (150 mM ammonium acetate, 300 mM sucrose, and distilled water) on the elemental content of cultured cells in suspension. The results demonstrated that distilled water was the best washing solution to prepare cultured cells. In addition, the low Na content, high K content and high K/Na ratio of the cells indicated that this procedure, based on the centrifugation at low g followed by cryopreparation, constitutes a satisfactory method to prepare cultured cells in suspension. We also investigated the effects of different accelerating voltages on X-ray signal collection. The results showed that moderate accelerating voltages, i.e. 10-11 kV, should be used to analyse whole cells in the scanning mode of the scanning electron microscope. We show that this method of preparation makes it possible to prepare cryosections of the cultured cells, thus permitting analysis of the elemental content at the subcellular level, i.e. nucleus, cytoplasm and mitochondria, using a scanning transmission electron microscope.
机译：我们描述了一个简单的程序来准备悬浮培养的细胞，以通过电子探针X射线显微分析在细胞水平上分析元素含量。将悬浮液中培养的细胞沉积在聚碳酸酯组织上，将培养板孔插入，以低g离心，洗涤以除去细胞外培养基，冷冻固定并冷冻干燥，并在扫描电子显微镜的扫描模式下进行分析。我们测试了不同洗涤溶液（150 mM醋酸铵，300 mM蔗糖和蒸馏水）对悬浮液中培养细胞元素含量的影响。结果表明，蒸馏水是制备培养细胞的最佳洗涤液。此外，细胞的低钠含量，高钾含量和高钾/钠比值表明，该方法基于低g离心再冷冻制备，是制备悬浮培养细胞的令人满意的方法。我们还研究了不同的加速电压对X射线信号收集的影响。结果表明，在扫描电子显微镜的扫描模式下，应使用中等的加速电压（10-11 kV）来分析整个细胞。我们表明，这种制备方法使制备培养细胞的冰冻切片成为可能，从而允许使用扫描透射电子显微镜分析亚细胞水平上的元素含量，即细胞核，细胞质和线粒体。

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