Flow-cytometric detection of changes in the fluorescence emission spectrum of a vital DNA-specific dye in human tumour cells

Abstract

A multiparameter flow cytometric technique has been used to detect changes in the emission spectrum of the DNA-specific fluorochrome Hoechst 33342 during uptake by intact, human tumour cells and during the in vitro titration of permeabilized cells. The spectral shift phenomenon was associated with changes in dye: DNA ratio revealing heterogeneity in dye-binding sites. The degree of spectral shift was sensitive to changes in pH within the physiological range. Surprisingly, chromatin structure, in terms of DNase accessibility, was not a major factor in the generation of the spectral shift. The technique of fluorescence emission analysis permits cells with similar DNA contents to be distinguished on the basis of changes in the microenvironment of chromatin for both fresh and freezer-stored biopsy or experimental preparations.