HIGH-AFFINITY BINDING OF 7-AMINOACTINOMYCIN-D AND 4',6-DIAMIDINO-2-PHENYLINDOLE TO HUMAN NEUTROPHILIC GRANULOCYTES AND LYMPHOCYTES

The binding behavior of the DNA binding dyes 7-aminoactinomycin D (7-A AMD) and 4',6-diamidino-2-phenylindole (DAPI) to human neutrophilic gr anulocytes and lymphocytes was studied by image cytofluorometry. Perip heral blood leukocytes were prefixed in paraformaldehyde (PEA) and att ached to cover glasses. Different fixation, permeabilization, and acid extraction methods were applied before the cells were stained to equi librium using varying concentrations of 7-AAMD or DAPI. The apparent a ssociation constant and number of high affinity dye binding sites were estimated for the different cell types, dyes, and treatments. Acid-ex tracted cells, supposedly containing nucleosome-free 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 d ye affinity was also reduced. We found no major difference in high aff inity binding between the cell types, but granulocytes showed more flu orescence from less specifically bound 7-AAMD compared to lymphocytes. DAPI had a much higher affinity than 7-AAMD, independent of the prepa ration method. It showed a cooperative binding behavior with an appare nt saturation of the high affinity binding sites at a dye concentratio n of about 50 nM. We conclude that both dyes may be useful as probes f or chromatin structure in intact cells and that our new technique may contribute to such studies since it allows determination of dye affini ties and numbers of high affinity binding sites in situ. (C) 1995 Wile y-Liss, Inc.