Detailed Studies on the Application of Three Fluorescent Antibiotics for Dna Staining in Flow Cytometry

Publisher Website
Google Scholar
Abstract
The effects of pH, ionic strength, stain concentration, Mg concentration and various fixative agents on DNA staining with the fluorescent antibiotics olivomycin, chromomycin A3 and mithramycin were examined with [calf thymus] DNA in solution and in mammalian [Chinese hamster ovary CHO] cells. Ethanol-fixed cells stained with mithramycin and analyzed by flow cytometry provided DNA distribution patterns with a high degree of resolution. Glutaraldehyde-fixed cells exhibited about 1/2 the fluorescence intensity of ethanol-fixed cells; the percentages of cells in G1, S and G2 + M were comparable. DNA distribution obtained for formalin-fixed cells were unacceptable for computer analysis. Cell staining over a pH range of 5-9 in solutions containing 0.15-1 M NaCl and 15-200 mM MgCl2 provided optimal results based on the DNA profiles obtained by flow cytometry. The intensity of cells stained in 1 M NaCl was 1.5 times greater than cells stained in the absence of NaCl; spectrophotofluorometric analysis of mithramycin-Mg DNA complexes in solution revealed no significant changes in fluorescence intensity over a range of 0-1.75 M NaCl. The increase in fluorescence of stained cells as a function of increasing ionic strength is apparently due to changes in chromatin structure, providing a larger number of binding sites for the dye-Mg complex.