High affinity binding of 7‐aminoactinomycin D and 4′,6‐diamidino‐2‐phenylindole to human neutrophilic granulocytes and lymphocytes

Abstract

The binding behavior of the DNA binding dyes 7-aminoactinomycin D (7-AAMD) and 4',6-diamidino-2-phenylindole (DAPI) to human neutrophilic granulocytes and lymphocytes was studied by image cytofluorometry. Peripheral blood leukocytes were prefixed in paraformaldehyde (PFA) and attached to cover glasses. Different fkation, permeabilization, and acid extraction methods were applied before the cells were stained to equilibrium using varying concentrations of 7-AAMD or DAPI. The apparent association constant and number of high afEnity dye binding sites were estimated for the different cell types, dyes, and treatments. Acidextracted cells, supposedly containing nucleosomefree DNA, were chosen to represent maximal dye binding. Only about 10% of the 7-AAMD binding sites remained in the unextracted PFA-fixed cells, and the apparent dye aiEnity was also reduced. We found no major difference in high a f h i t y binding between the cell types, but granulocytes showed more fluorescence from less specitically bound 7-AAMD compared to lymphocytes. DAPI had a much higher alfinity than 7-AAMD, independent of the preparation method. It showed a cooperative binding behavior with an apparent saturation of the high afhity binding sites at a dye concentration of about 50 nM. We conclude that both dyes may be useful as probes for chromatin structure in intact cells and that our new technique may contribute to such studies since it allows determination of dye aEdties and numbers of high aSanity binding sites ill S h . 0 1995 Wiley-Liss, Inc.

Accessibility of 7-aminoactinomycin D to lymphocyte nuclei after paraformaldehyde fixation

“…A decrease in fluorescence intensity can be explained by a decrease in the number of 7-AAMD binding sites or a reduced affinity. However, previous results have shown that the affinity constant was not affected by cell preparations unless nuclear proteins were removed (9). Therefore, the decrease in fluorescence intensity after PFA fixation is most probably an effect of a decrease in the number of available dye binding sites.…”

“…7-AAMD accessibility to cultured fibroblasts has been shown to decrease with about 50% after PFA fixation (14), and we have also shown the importance of distinguishing between specific, high affinity binding, and non-specific dye binding (9). In this study we have used 7-AAMD to investigate the dynamics of PFA crosslinking of nuclear proteins to DNA.…”

Affinity of linker histones for chromatin in situ analyzed using DAPI as a cytochemical probe

“…Human granulocytes were isolated from whole blood obtained from healthy donors and prepared as described previously (27).…”

DNA binding fluorochromes as probes for histone H1-chromatin interactions in situ

“…Many DNA binding fluorochromes show restrictions in their binding to DNA by the chromatin structure, and extraction of basic proteins results in an increased nuclear fluorescence intensity (11,12,15,19,21). Salt extraction of H1 also results in higher fluorescence intensity (15,23).…”

“…An obvious candidate for the study of protein-DNA interactions is the DNA binding fluorochrome 7-aminoactinomycin D (7-AAMD), which is regarded as rather sensitive to such interactions (12,21,24). Actinomycin D may have some preference for active chromatin (3,34), where the amount of linker histones may be reduced.…”