Quantitating the concentration of Py-Im polyamide-fluorescein conjugates in live cells.
Quantitative fluorescence-based methods have been developed to determine the nuclear concentration of polyamide-fluorescein conjugates in cell culture. Confocal laser scanning microscopy and flow cytometry techniques are utilized to plot calibration curves, from which the nuclear concentration can be interpolated. Upon treatment with polyamide, the concentration in the nucleus of live HeLa cells is calculated to be between 0.1 and 0.5microM, which is significantly lower than the 2microM dosage concentration. In contrast, the observed nuclear concentration in U251 cells is closer to the dosage concentration, indicating a cell line-specific increase in uptake for this class of compounds. Although confocal microscopy and flow cytometry generate disparate values, taken together these experiments suggest that the polyamide concentration inside the cell nucleus is lower than it is outside the cell.
Hsu
Carey F
CF
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
Dervan
Peter B
PB
D000818:Animals; D002417:Cattle; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000737:chemistry; D004247:DNA / Q000032:analysis; D005434:Flow Cytometry / Q000379:methods; D019793:Fluorescein / Q000032:analysis*; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006367:HeLa Cells; D006801:Humans; D007093:Imidazoles / Q000032:analysis*; D018613:Microscopy, Confocal / Q000379:methods; D009757:Nylons / Q000032:analysis*; D011758:Pyrroles / Q000032:analysis*; D021241:Spectrometry, Mass, Electrospray Ionization; D013950:Thymus Gland / Q000737:chemistry
D005456:Fluorescent Dyes; D007093:Imidazoles; D009757:Nylons; D011758:Pyrroles; D004247:DNA; D016650:Fluorescein-5-isothiocyanate; D019793:Fluorescein