A procedure to prepare cultured cells in suspension for electron probe X-ray microanalysis: application to scanning and transmission electron microscopy.
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.
Fernández-Segura
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Department of Cell Biology, School of Medicine, University of Granada, E-18071 Granada, Spain. efernandez@histolii.ugr.es
Cañizares
F J
FJ
Cubero
M A
MA
Campos
A
A
Warley
A
A
D002451:Cell Compartmentation; D015925:Cryopreservation / Q000379:methods*; D004577:Electron Probe Microanalysis / Q000379:methods*; D006801:Humans; D008854:Microscopy, Electron / Q000379:methods*; D008855:Microscopy, Electron, Scanning / Q000379:methods; D020298:U937 Cells / Q000737:chemistry* / Q000648:ultrastructure