Spectral studies on 33258 Hoechst and related bisbenzimidazole dyes useful for fluorescent detection of deoxyribonucleic acid synthesis.

Latt, Samuel A.; Stetten, Gail

doi:10.1177/24.1.943439

Cited by 342 publications

(172 citation statements)

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“…Incubation of cells in the presence of BrdUrd results in incorporation of this precursor into DNA resulting in altered staining properties of this biopolymer (11)(12)(13)(14)(15). In a previous paper we reported that after differential staining of DNA versus RNA with AO, proliferating human lymphocytes exposed to BrdUrd for one generation had a 40% decrease in DNase-sensitive green fluorescence when compared with noncycling lymphocytes or cycling cells incubated in the absence of this precursor (4).…”

Section: Discussionmentioning

confidence: 99%

“…The present technique, as well as other techniques of BrdUrd detection, based on suppression or enhancement of DNA stainability with various fluorochromes (1,4, [11][12][13][14][15]17) suffer in general from low sensitivity. In this respect they are inferior to immunochemical approach (9) which detects as little as 4% of the replicated genome and, therefore, may be applied after short (30 min) pulse of BrdUrd.…”

Section: Discussionmentioning

confidence: 99%

“…The BrdUrd-attributed suppression of cell fluorescence is large enough to separate totally BrdUrd labeled from unlabeled mitotic populations. The combination of these two flow cytometric techniques, one which allows discrimination between interphase and metaphase cells and the other which allows further subdivision of metaphase cells into those which have incorporated BrdUrd and those which have not, provides an alternative to the autoradiographic procedure necessary for obtaining the fraction of labeled mitoses.Key terms: Flow cytometry, acridine orange, DNA replication, cell cycle analysis, fluorescence quenching, bromodeoxyuridine incorporationAs an alternative to thymidine autoradiography, several flow cytometric techniques have been designed to quantify cells incorporating the thymidine analog 5-bromodeoxyuridine (BrdUrd) (1,4,9,(12)(13)(14)(15)17). Detection of cells incorporating this precursor is based either on immunochemical procedures utilizing BrdUrd specific antibodies (9) or on the BrdUrd induced quenching of fluorescence of such dyes as acridine orange (4) or Hoechst 33258 (1,(11)(12)(13)(14)(15).…”

mentioning

confidence: 99%

“…Detection of cells incorporating this precursor is based either on immunochemical procedures utilizing BrdUrd specific antibodies (9) or on the BrdUrd induced quenching of fluorescence of such dyes as acridine orange (4) or Hoechst 33258 (1,(11)(12)(13)(14)(15). BrdUrd enhanced fluorescence of the DNA bound dye mithramycin also provides a basis for flow cytometric recognition of cells synthesizing DNA (17).…”

mentioning

confidence: 99%

“…As an alternative to thymidine autoradiography, several flow cytometric techniques have been designed to quantify cells incorporating the thymidine analog 5-bromodeoxyuridine (BrdUrd) (1,4,9,(12)(13)(14)(15)17). Detection of cells incorporating this precursor is based either on immunochemical procedures utilizing BrdUrd specific antibodies (9) or on the BrdUrd induced quenching of fluorescence of such dyes as acridine orange (4) or Hoechst 33258 (1,(11)(12)(13)(14)(15).…”

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Distinction between 5‐bromodeoxyuridine labeled and unlabeled mitotic cells by flow cytometry

1983

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Exponentially growing Friend leukemia cells are exposed to 5-bromodeoxyuridine (BrdUrd) for the time period equivalent to one generation. After fixation and incubation with RNase, DNA in situ is partially denatured by acid and the cells are stained with acridine orange (AO). Staining with A 0 under these conditions reveals the extent of denatured versus double stranded DNA and enables one to distinguish mitotic from interphase cells. It is observed that all BrdUrd treated cells, regardless of cell cycle phase, have decreased both green and red fluorescence. This finding suggests that BrdUrd interacts with A 0 not only in the complexes of the dye with double stranded DNA, but also with the single stranded biopolymer. The BrdUrd-attributed suppression of cell fluorescence is large enough to separate totally BrdUrd labeled from unlabeled mitotic populations. The combination of these two flow cytometric techniques, one which allows discrimination between interphase and metaphase cells and the other which allows further subdivision of metaphase cells into those which have incorporated BrdUrd and those which have not, provides an alternative to the autoradiographic procedure necessary for obtaining the fraction of labeled mitoses.Key terms: Flow cytometry, acridine orange, DNA replication, cell cycle analysis, fluorescence quenching, bromodeoxyuridine incorporationAs an alternative to thymidine autoradiography, several flow cytometric techniques have been designed to quantify cells incorporating the thymidine analog 5-bromodeoxyuridine (BrdUrd) (1,4,9,(12)(13)(14)(15)17). Detection of cells incorporating this precursor is based either on immunochemical procedures utilizing BrdUrd specific antibodies (9) or on the BrdUrd induced quenching of fluorescence of such dyes as acridine orange (4) or Hoechst 33258 (1,(11)(12)(13)(14)(15). BrdUrd enhanced fluorescence of the DNA bound dye mithramycin also provides a basis for flow cytometric recognition of cells synthesizing DNA (17).In earlier studies, we demonstrated that cells in mitosis may be distinguished from interphase cells by flow cytometry, based on differences in chromatin structure (3, 7, 8). Specifically, after enzymatic removal of RNA and partial DNA denaturation in situ by heat or acid (7) (ds) versus denatured DNA was achieved by using the metachromatic fluorochrome, acridine orange (AO) (5). In the technique we describe here, partial denaturation of DNA in situ is combined with BrdUrd induced quenching of A 0 to identify and count differentially BrdUrd labeled mitotic cells. The cells in cultures are grown for approximately one generation in the presence of BrdUrd. After staining with AO, both green and red fluorescence of BrdUrd labeled cells are suppressed regardless of the phase of the cell cycle. The differences in fluorescence intensity are large enough to obtain total separation of BrdUrd labeled and unlabeled mitotic cells. Materials and MethodsCells: Friend leukemia (FL) cells, strain 745, were obtained from the Medical Research Institute, Camden, New ...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Distinction between 5‐bromodeoxyuridine labeled and unlabeled mitotic cells by flow cytometry

1983

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DNA structural features responsible for sequence-dependent binding geometries of Hoechst 33258

et al. 1998

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Nucleic acid specificity of an acridine derivative permits its use for flow cytometric analysis of the cell cycle

Jayat‐Vignoles

Ratinaud²

1997

Cytometry

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3‐amino‐6‐methoxy‐9‐(2‐hydroxyethylamine) acridine (AMHA) is an acridine derivative, which is easily excited in near ultraviolet and which emits a bright green fluorescence. The dye was preferentially incorporated into nucleic structures as attested by microscopic and cytometric analyses after RNase and DNase treatments. The affinity for RNA seemed low and similar to that observed for propidium iodide. AMHA was quickly accumulated in fixed cells, and in appropriate concentrations (10–50 μM) was a DNA‐ and RNA‐specific dye. AMHA probably exhibits an adenine‐thymine specificity, as suggested by its quenching after bromodeoxyuridine uptake: the fluorescence quenching was similar to that obtained for Hoechst 33258. After cell treatment by RNase and in the presence of MgCl2, AMHA staining allowed flow cytometric analysis of the cell‐cycle distribution. The resulting histograms were similar to those obtained with propidium iodide (CV near 3.5%, and similar cell cycle distribution). Thus, AMHA is a suitable fluorescent dye for efficient analysis of the cell cycle by flow cytometry. Cytometry 27:153–160, 1997. © 1997 Wiley‐Liss, Inc.

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Spectral studies on 33258 Hoechst and related bisbenzimidazole dyes useful for fluorescent detection of deoxyribonucleic acid synthesis.

Cited by 342 publications

References 24 publications

Distinction between 5‐bromodeoxyuridine labeled and unlabeled mitotic cells by flow cytometry

Distinction between 5‐bromodeoxyuridine labeled and unlabeled mitotic cells by flow cytometry

DNA structural features responsible for sequence-dependent binding geometries of Hoechst 33258

Nucleic acid specificity of an acridine derivative permits its use for flow cytometric analysis of the cell cycle

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