Characteristics of a novel deep red/infrared fluorescent cell-permeant DNA probe, DRAQ5, in intact human cells analyzed by flow cytometry, confocal and multiphoton microscopy

Smith, Paul J.; Blunt, Nicola; Wiltshire, Marie; Hoy, Terry; Teesdale‐Spittle, Paul; Craven, MT; Watson, James V.; Amos, W. B.; Errington, Rachel J.; Patterson, LH

doi:10.1002/1097-0320(20000801)40:4<280::aid-cyto4>3.0.co;2-7

Cited by 151 publications

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“…The anticancer agent mitoxantrone and modified anthraquinones show variable DNA-affinic and cell-permeation properties (5,(24)(25)(26)(27), with the probe DRAQ5 readily crossing the plasma membrane of intact living cells (5,6). The current study on this 1,5-bisalkylaminoanthraquinone has addressed target binding and spectral properties in live cells.…”

Section: Discussionmentioning

confidence: 99%

“…Such considerations provide a clear impetus for understanding the information content of fluorescence signatures from single cells. Here, we have undertaken spectral analysis of the behavior of the novel DNA binding agent, DRAQ5 (5,6), in intact cells to address binding modes in live cells, nuclear feature resolution, and cell cycle profiling, with specific reference to the application of GFP-based reporters.…”

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Spectral analysis of the DNA targeting bisalkylaminoanthraquinone DRAQ5 in intact living cells

et al. 2006

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Background: We report on the potential DNA binding modes and spectral characteristics of the cell-permeant far red fluorescent DNA dye, DRAQ5, in solution and bound within intact cells. Our aim was to determine the constraints for its use in flow cytometry and bioimaging. Methods: Solution characteristics and quantum yields were determined by spectroscopy. DRAQ5 binding to nuclear DNA was analyzed using fluorescence quenching of Hoechst 33342 dye, emission profiling by flow cytometry, and spectral confocal laser scanning microscopy of the complex DRAQ5 emission spectrum. Cell cycle profiling utilized an EGFP-cyclin B1 reporter as an independent marker of cell age. Molecular modeling was used to explore the modes of DNA binding. Results: DRAQ5 showed a low quantum yield in solution and a spectral shift upon DNA binding, but no significant fluorescence enhancement. DRAQ5 caused a reduction in the fluorescence intensity of Hoechst 33342 in live cells prelabeled with the UV excitable dye, consistent with molecular modeling that suggests AT preference and an en-

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Spectral analysis of the DNA targeting bisalkylaminoanthraquinone DRAQ5 in intact living cells

et al. 2006

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“…In view of these caveats of the Hoechst/DAPI DNA dye family, we tested the recently described DNA intercalator DRAQ5 (12,13). The comparison of DRAQ5 nuclear DNA staining with H2B-GFP-labeled chromatin in live cells showed a costaining of the same nuclear regions and the chromosomes in mitotic cells (Fig.…”

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DNA labeling in living cells

2005

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Background: Live cell fluorescence microscopy experiments often require visualization of the nucleus and the chromatin to determine the nuclear morphology or the localization of nuclear compartments. Methods: We compared five different DNA dyes, TOPRO-3, TOTO-3, propidium iodide, Hoechst 33258, and DRAQ5, to test their usefulness in live cell experiments with continuous imaging and photobleaching in widefield epifluorescence and confocal laser scanning microscopy. In addition, we compared the DNA stainings with fluorescent histones as an independent fluorescent label to mark chromatin.Results: From the dyes tested, only Hoechst and DRAQ5 could be used to stain DNA in living cells. However,

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“…We chose the novel deep red fluorescent dye Draq5 instead of the more commonly used propidium iodide as DNA dye and trigger parameter, since it displays less spectral overlap with the emission spectrum of Alexa488 (Figs. 1A and 1B; green integral) (29), thereby avoiding the need for electronic signal compensation during the measurement of HIF-1a based on the second fluorophore. Conversely, Draq5 can be excited with the HeNe laser and its emission can therefore be measured during the second scan step separated from the Argon laser, which avoids spectral overlap that may arise from Alexa488.…”

Section: Measurement Of Hif-1a In Two Cervical Carcinomamentioning

confidence: 99%

Analysis of hypoxia‐inducible factor‐1α accumulation and cell cycle in geldanamycin‐treated human cervical carcinoma cells by laser scanning cytometry

2005

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Background: Tumor hypoxia has been linked to increased disease aggressiveness and poorer treatment outcomes, and the transcription factor hypoxia-inducible factor-1 (HIF-1) has been identified as the key molecule mediating the cellular response to hypoxic microenvironments. The a-subunit of this factor is accumulated under hypoxia and rapidly degraded during re-oxygenation, rendering the reliable measurement of HIF-1a a difficult task. Heat shock protein 90 (Hsp90) is an essential protein that controls the activity, turnover, and trafficking of a variety of other proteins including HIF-1a and cell cycle regulators. Hsp90 inhibitors like geldanamycin therefore have the potential to target tumor-cell survival by at least two mechanisms, compromising the accumulation of HIF-1a and cell proliferation. Methods: We describe here the simultaneous measurement of HIF-1a and cell cycle parameters by laser scanning cytometry (LSC) after exposure of two different

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Characteristics of a novel deep red/infrared fluorescent cell-permeant DNA probe, DRAQ5, in intact human cells analyzed by flow cytometry, confocal and multiphoton microscopy

Cited by 151 publications

References 20 publications

Spectral analysis of the DNA targeting bisalkylaminoanthraquinone DRAQ5 in intact living cells

Spectral analysis of the DNA targeting bisalkylaminoanthraquinone DRAQ5 in intact living cells

DNA labeling in living cells

Analysis of hypoxia‐inducible factor‐1α accumulation and cell cycle in geldanamycin‐treated human cervical carcinoma cells by laser scanning cytometry

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