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Comparison of reagents for shape analysis of fixed cells by automated fluorescence microscopy
John T Elliott1, Alessandro Tona, Anne L Plant
1Biomolecular Materials Group, Biotechnology Division, Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, USA. jelliot@nist.gov
Background:
Cell size and shape have been implicated as potentiators of intracellular signaling events and as indicators of abnormal cell behavior. Automated microscopy and image analysis can provide quantitative information about the size and shape of cultured cells, but it requires that the edge of a cell be clearly identified. Generating adequate contrast at the edge of thin well-spread cells can be challenging.
Methods:
We compared six (five chemically reactive and one lipophilic) fluorescent molecules--5-chloromethyl fluorescein diacetate (CMFDA, CellTracker green), fluorescein-5-maleimide, fluorescein-5-isothiocyanate (FITC), 5-iodoacetamidofluorescein, 5(6)-carboxy fluorescein-N-hydroxysuccinimidyl ester, and N-fluorescein-1,2-dihexadecanoyl-sn-glycerol-3-phosphoethanolamine--for their effectiveness as stains for automated morphology analysis of fixed cells.
Results:
Formaldehyde-fixed rat aortic smooth muscle cells stained with fluorescein-5-maleimide or FITC exhibited an average intensity that was at least twofold greater than cells stained with CMFDA even when subjected to a 25-fold shorter exposure time. Cell area determined with the higher intensity stains was less sensitive to threshold settings during automated cell morphology analysis.
Conclusion:
A procedure that includes the use of fluorescein-5-maleimide or FITC for staining fixed cell provides sensitivity sufficient to permit rapid, automated, morphologic analysis of well-spread fixed cells.