Distinction of leucocyte classes based on chromatin-structure-dependent DNA-binding of 7-aminoactinomycin D.
Binding of the DNA-specific dye 7-aminoactinomycin D (7-AMD) in chromatin of human leucocytes was studied by flow cytometry. After formaldehyde fixation and permeabilization, monocytes bound 30-130% more 7-AMD than lymphocytes, while binding in granulocytes was 20-60% higher than in lymphocytes. Monocytes and lymphocytes bound similar amounts of 7-AMD when cells were permeabilized by detergent prior to fixation. Digestion of DNA in formaldehyde-fixed chromatin by DNase 1 was quantitated by measuring Hoechst 33258 (H33258) fluorescence of mononuclear cells. The monocyte/lymphocyte H33258 fluorescence ratio decreased with DNase 1 digestion to an asymptotic value of 0.74, showing that DNA in chromatin of monocytes was more susceptible to DNase 1 digestion. 7-AMD binding increased, reached a maximum and then decreased with extent of DNase 1 digestion in both mononuclear cell types. The monocyte/lymphocyte 7-AMD fluorescence ratio also decreased after DNase 1 digestion. RNA content and RNA synthesis were higher in monocytes than in lymphocytes. The results show that 7-AMD binding in chromatin of mononuclear leucocytes correlates with transcriptional activity as measured by DNase1 susceptibility and RNA synthesis. The staining procedure may be used for differential counting of mature myeloid cells in peripheral blood and bone marrow.
D000911:Antibodies, Monoclonal; D002843:Chromatin / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D003609:Dactinomycin / Q000031:analogs & derivatives*; D003850:Deoxyribonuclease I; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006801:Humans; D007962:Leukocytes / Q000145:classification*; D013194:Staining and Labeling; D014158:Transcription, Genetic
D000911:Antibodies, Monoclonal; D002843:Chromatin; D005456:Fluorescent Dyes; D003609:Dactinomycin; C025942:7-aminoactinomycin D; D004247:DNA; D003850:Deoxyribonuclease I