7‐amino‐actinomycin D as a specific fluorophore for DNA content analysis by laser flow cytometry

Zelenin, A. V.; Poletaev, A. I.; Stepanova, Natalija G.; Barsky, V. E.; Kolesnikov, V. A.; Nikitin, S.; Zhuze, A. L.; Gnutchev, Nikolai V.

doi:10.1002/cyto.990050410

Cited by 50 publications

(29 citation statements)

References 19 publications

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“…The need for a second laser is only a minor disadvantage, since there are many excellent research flow cytometers which are equipped as standard with a low-power HeNe laser, for instance the Coulter Elite flow cytometer (19). 7AAD and Hoechst 33342 have been proposed as alternatives for PI in multiparameter flow cytometry (1,11,14,20). However, in our hands, using epithelial cells, 7AAD gave unacceptable high coefficients of variation (CVs) (data not shown).…”

Section: Resultsmentioning

confidence: 63%

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TO‐PRO‐3 iodide: A novel HeNe laser‐excitable DNA stain as an alternative for propidium iodide in multiparameter flow cytometry

1994

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A new red emitting fluorophore, TO-PRO-3 iodide (TP3), which is best excited by an HeNe laser (633 nm), has been compared with propidium iodide (PI) for measuring relative DNA content. TP3, which has a peak absorbance at 642 nm and emission at 661 nm, has been tested on peripheral blood lymphocytes (PBL) and keratinocytes in a two-laser system. As an example, we present a three-color flow cytometric application utilizing TP3 in combination with fluorescein-isothiocyanate (FITC) and phycoerythrin (PE) conjugated to monoclonal antibodies in this paper.A subequilibrium concentration of 1 pM TP3, most preferably used in combination with RNase treatment, proved to be a powerful alternative for DNA amount determination. In humanand mouse-Balb/MK-keratinocyte populations with different S-phase fractions, PI and TP3 showed a good correlation.Finally, in the triple labelling experiment we clearly demonstrated that TP3 is readily applied to the analysis of binding of two antibodies and relative DNA content simultaneously. 0 1994 Wiley-Liss, Inc.

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Section: Resultsmentioning

confidence: 63%

“…In 1984, Zelenin et al investigated an alternative DNA stain, 7-amino-actinomycin D (7AAD), which has an emission spectrum with a maximum at a longer wavelength compared to PI (>650 nm) (20). Although the quantum efficiency is low, it can be used in combination with FITC-and PE-labeled antibodies using only one single (488 nm) excitation beam (11).…”

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confidence: 99%

TO‐PRO‐3 iodide: A novel HeNe laser‐excitable DNA stain as an alternative for propidium iodide in multiparameter flow cytometry

1994

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“…Cells stained in the presence of Triton X-100 were permeable to both dyes; similar results were obtained for staining times from 30 minutes to 1 week. The H33258 concentration must be kept low to avoid secondary binding (371, while it appears that the 7-AMD concentration is not critical (42). H33258 did not compete for 7-AMD binding sites, since similar results (546-nm excitation) were obtained when H33258 was omitted from the staining buffer.…”

Section: Stainingmentioning

confidence: 89%

Distinction of leucocyte classes based on chromatin‐structure‐dependent DNA‐binding of 7‐aminoactinomycin D

Stokke

Steen

1987

Cytometry

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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 20-6096 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 DNasel was quantitated by measuring Hoechst 33258 (H33258) fluorescence of mononuclear cells. The monocyte/ lymphocyte H33258 fluorescence ratio decreased with DNasel digestion to an asymptotic value of 0.74, showing that DNA in chromatin of monocytes was more susceptible to DNasel digestion. 7-AMD binding increased, reached a maximum and then decreased with extent of DNasel digestion in both mononuclear cell types. The monocyte/ lymphocyte 7-AMD fluorescence ratio also decreased after DNasel 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 DNasel susceptibility and RNA synthesis. The staining procedure may be used for differential counting of mature myeloid cells in peripheral blood and bone marrow.

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“…Owing to the complex structure of the molecule it is difficult to predict its precise interaction with biological membranes at a given pH; its size however will impede membrane penetration. 7-AAD forms very stable complexes with DNA (15). Its very high binding constant and slow dissociation rate (6) allow the use of 7-AAD at low dye concentration which reduces the amount of unbound free dye that could attach passively to the surface of live cells and could eventually lead to staining of live cells.…”

Section: Discussionmentioning

confidence: 99%

“…7-amino-actinomycin D (7-AAD) is a G-C base-specific DNA intercalator (1) that can be excited by the ' 488 nm argon laser line (15) and emits in the far red range of the spectrum (Fig. 1); 7-AAD spectral emission can be effectively separated from the emissions of FITC and PE.…”

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confidence: 99%

Dead cell discrimination with 7‐amino‐actinomcin D in combination with dual color immunofluorescence in single laser flow cytometry

Krall²,

et al. 1992

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Identification of nonviable cells in immunofluorescently stained cell populations is essential for obtaining accurate data. Fluorescent non‐vital DNA dyes, particularly propidium iodide (PI), have been used routinely in flow cytometry for discrimination of dead cells from viable cells on the basis of fluorescence. We describe here the use of an alternative DNA dye, 7‐amino‐actinomycin D (7‐AAD), which can replace PI for the exclusion of nonviable cells. As an example, we present in this paper the utilization of 7‐AAD on various leukemic cell lines for dead cell exclusion whenever the viable cell population could not be discriminated reliably from nonviable cells on the light scatter histogram; 7‐AAD is suitable for dead cell discrimination in lengthy experiments because it is efficiently excluded by intact cells and has a high DNA binding constant. In addition, the dye is valuable in combination with phycoerythrin (PE)‐fluorescence dual‐color flow cytometry on a single argon laser instrument, since its emission in the far red can easily be separated from the emission of PE; 7‐AAD was used on fluoresceinisothiocyanate (FITC) and PE surface‐labeled human thymocytes for characterization of the dying subpopulation of cells which is undergoing programmed cell death. In this heterogeneous cell preparation, the spectral properties of the dye permitted the classification of viable and nonviable cell subpopulations by multiparameter analysis.

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7‐amino‐actinomycin D as a specific fluorophore for DNA content analysis by laser flow cytometry

Cited by 50 publications

References 19 publications

TO‐PRO‐3 iodide: A novel HeNe laser‐excitable DNA stain as an alternative for propidium iodide in multiparameter flow cytometry

TO‐PRO‐3 iodide: A novel HeNe laser‐excitable DNA stain as an alternative for propidium iodide in multiparameter flow cytometry

Distinction of leucocyte classes based on chromatin‐structure‐dependent DNA‐binding of 7‐aminoactinomycin D

Dead cell discrimination with 7‐amino‐actinomcin D in combination with dual color immunofluorescence in single laser flow cytometry

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