Re-evaluation of acridine orange stain for flow cytometry

Laser flow measurements of scattering and fluorescence from cell nuclei in the presence of increasing Mg++ concentrations

“…The process of AO intercalation can be considered a simulation of the accessibility of the genome to biological molecules under physiological conditions. As already indicated in the literature (Traganos et al, 1976;Beltrame et al, 1981), changes in Mg"+ concentrations can affect the intercalation process by modifying the number of available binding sites (while interfering with the stacking process by modifying its equilibrium constant). A decrease in the fluorescence intensity (green emission-intercalation process) can be assumed, as a first approximation, to indicate a reduction in the availability of binding sites.…”

“…For this second set of experiments, the fluorescent dye acridine orange (AO) was used as a probe for the DNA amount and the overall chromatin accessibility. The dye is known to intercalate (as a cation) in monomeric form between the DNA base pairs, while the experimental conditions used (2 * 10-5 M final AO concentration and 3 -105 nuclei/ml) should guarantee the absence of undesired secondary processes (e.g., stacking) Beltrame et al, 1981). Because the intercalation process results in the emission of green fluorescent light, whereas the stacking process induces a shift to longer wavelengths (orange-red), a qualitative control of the staining procedure can be easily performed by visual inspection with a fluorescence microscope; our experimental conditions were periodically checked in this way.…”

“…Measurements of the fluorescence intensity resulting from the intercalation of dyes between the DNA base pairs are frequently performed to obtain information about DNA accessibility in chromatin under various physiological conditions. This approach is quite a sensitive one, even if the control of all the staining steps is rather difficult Beltrame et al, 1981).…”