Towards microfluorometric quantitation of polyamines in situ. Relationship between cellular polyamine concentration and fluorescence yield of the formaldehyde fluorescamine method.
Two different fluorescence cytochemical methods, the formaldehyde-fluorescamine (FF) method and the orthophthalaldehyde (OPT) method as well as an immunocytochemical method have been developed for the localization of spermidine and spermine. Of these three methods, the FF-method is the most easy to perform. We have studied the relationship between fluorescence intensity induced by the FF-method and cellular polyamine levels measured by HPLC in MCF-7 cells and HeLa cells. The experiments were designed to obtain different cell concentrations of polyamines. Cells grown on microscope slides in Petri-dishes were partly depleted of spermidine by two days inhibition of their ornithine decarboxylase activity using alpha-difluoromethylornithine. One hr before harvest the cells were exposed to different concentrations (0-30 microM) of spermidine. Microfluorometric results and chemical determinations of spermidine and spermine were obtained from each separate slide. The cellular total polyamine (spermidine + spermine) concentration on the slides varied between 4 and 15 nmol per mg protein (MCF-7 cells) and 5 and 26 nmol per mg protein (HeLa cells) and the corresponding microfluorometric results between 60 and 115 arbitrary units (MCF-7 cells) and 80 and 160 arbitrary units (HeLa cells). Simple regression analysis showed a good linear relationship between cellular polyamine concentration and FF-fluorescence yield. The correlation coefficient for MCF-7 cells was 0.86 and for HeLa cells 0.82, significance of the correlations was p less than or equal to 0.0001. Our results add further credence to the specificity of the FF-method and indicate that the method may be useful for microfluorometric quantitation of polyamines in situ.
Hougaard
D M
DM
Department of Molecular Cell Biology, State Serum Institute, Copenhagen, Denmark.
Larsson
L I
LI
D001943:Breast Neoplasms / Q000032:analysis / Q000473:pathology; D003592:Cytophotometry / Q000379:methods; D005450:Fluorescamine; D005453:Fluorescence; D005557:Formaldehyde; D006367:HeLa Cells / Q000032:analysis / Q000473:pathology; D006801:Humans; D008722:Methods; D013095:Spermidine / Q000032:analysis*; D013096:Spermine / Q000032:analysis*; D014407:Tumor Cells, Cultured; D009764:o-Phthalaldehyde
D005557:Formaldehyde; D013096:Spermine; D005450:Fluorescamine; D009764:o-Phthalaldehyde; D013095:Spermidine