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

被引:9
作者
LOBORG, H [1 ]
LINDEN, E [1 ]
LONN, A [1 ]
SKOGLUND, P [1 ]
RUNDQUIST, I [1 ]
机构
[1] LINKOPING UNIV,FAC HLTH SCI,DEPT CELL BIOL,S-58185 LINKOPING,SWEDEN
来源
CYTOMETRY | 1995年 / 20卷 / 04期
关键词
DNA; CHROMATIN STRUCTURE; DYE BINDING; HUMAN LEUKOCYTES;
D O I
10.1002/cyto.990200405
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 (PEA) and attached to cover glasses. Different fixation, 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 affinity dye binding sites were estimated for the different cell types, dyes, and treatments. Acid-extracted 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 dye affinity was also reduced. We found no major difference in high affinity binding between the cell types, but granulocytes showed more fluorescence from less specifically bound 7-AAMD compared to lymphocytes. DAPI had a much higher affinity than 7-AAMD, independent of the preparation method. It showed a cooperative binding behavior with an apparent saturation of the high affinity 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 affinities and numbers of high affinity binding sites in situ. (C) 1995 Wiley-Liss, Inc.
引用
收藏
页码:296 / 306
页数:11
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