Document Detail


Preparation of cells cultured on silicon wafers for mass spectrometry analysis.
MedLine Citation:
PMID:  15940684     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The distribution of specific atoms and molecules within living cells is of high interest in bio-medical research. Laser secondary neutral mass spectrometry (laser-SNMS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS) detect atoms with high sensitivity and spatial resolution. The application of these methods to cultured cells requires special preparation techniques preserving morphological and chemical integrity of the living cells. The cells should, therefore, be grown on a conducting material preventing charging of the sample during ion bombardment. Silicon is currently used as the preferred support material for non-biological samples in mass spectrometry. This study investigates (1) the influence of silicon surfaces on cell growth and (2) the suitability of a sandwiched, rapid freezing method to analyse transmembrane ion gradients. Human melanoma cells were grown on silicon with polished or etched surfaces. Growth kinetics were studied using the Sulforhodamine-B assay. Number, shape, and morphology of the cells were assessed by epifluorescence microscopy of calcein AM- and DAPI-stained cells. Cells were subjected to rapid freezing, freeze-fracturing, and freeze-drying prior to analysis by TOF-SIMS and laser-SNMS. While cell numbers and morphology on the rough silicon wafers were impaired, morphology and growth kinetics of cells on polished silicon were identical to control cells on cell culture tested polystyrene. TOF-SIMS and laser-SNMS resulted in high-resolution elemental images and mass spectra. Measurement of the intracellular Na+ and K+ concentrations revealed a ratio as observed in living cells. In conclusion, culturing cells on polished silicon wafers followed by sandwiched, rapid freezing is an adequate preparation method to study intracellular ion distribution with mass spectrometry.
Authors:
Andrea Wittig; Martin Wiemann; Michael Fartmann; Christian Kriegeskotte; Heinrich F Arlinghaus; Karl Zierold; Wolfgang Sauerwein
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Publication Detail:
Type:  Comparative Study; Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Microscopy research and technique     Volume:  66     ISSN:  1059-910X     ISO Abbreviation:  Microsc. Res. Tech.     Publication Date:  2005 Apr 
Date Detail:
Created Date:  2005-06-13     Completed Date:  2005-08-01     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  9203012     Medline TA:  Microsc Res Tech     Country:  United States    
Other Details:
Languages:  eng     Pagination:  248-58     Citation Subset:  IM    
Copyright Information:
(c) 2005 Wiley-Liss, Inc.
Affiliation:
Strahlenklinik, Universität Duisburg-Essen, 45122 Essen, Germany. andrea.wittig@uni-essen.de
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MeSH Terms
Descriptor/Qualifier:
Cell Culture Techniques / methods*
Cell Line, Tumor
Cryopreservation
Freezing
Humans
Mass Spectrometry / methods*
Silicones
Chemical
Reg. No./Substance:
0/Silicones

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