<scp>DRAQ</scp>5 Labeling of Nuclear <scp>DNA</scp> in Live and Fixed Cells

Smith, Paul J.; Wiltshire, Marie; Errington, Rachel J.

doi:10.1002/0471142956.cy0725s28

Cited by 36 publications

(31 citation statements)

References 26 publications

(47 reference statements)

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“…8,9,14,37 When bound to DNA, in contrast to PicoGreen, we observed a continuous increase in the contour length, starting from the lowest dye concentration (DRAQ5:bp = 0.29:1). These results support that DRAQ5 acts as a strong intercalator, since intercalation leads to DNA base unwinding and elongation.…”

Section: ■ Conclusionmentioning

confidence: 78%

“…Interestingly, this dye is often used for live cell imaging. 8,9,14,37 Its excitation maximum is at 646 nm and emission spectra at 697 nm.…”

Section: ■ Resultsmentioning

confidence: 99%

“…The need to label and visualize DNA and chromatin is gaining importance in different fields of biological research, with experiments ranging from measurements on isolated DNA 1−6,11−13 to experiments in fixed cells and live cells. [7][8][9][10]14 There is a large variety of DNA labeling dyes: some binding to the minor/major grooves of double-stranded DNA, others intercalating into DNA, and some doing both. 1,8−20 The influence of DNA labeling dyes on the DNA structure and its properties is still a topic of debate, since no clear data exist on how strongly they influence physical properties.…”

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confidence: 99%

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Influence of DNA Binding Dyes on Bare DNA Structure Studied with Atomic Force Microscopy

et al. 2015

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Fluorescent dyes are widely used for staining and visualization of DNA in optical microscopy based methods. Even though for some dyes the mechanism of binding is known, how this binding affects DNA remains poorly understood. Here we present a novel experimental study of the influence of staining dyes on DNA properties. We use atomic force microscopy which allows quantification and measurement of structural properties of stained DNA with nanometer resolution. We studied the influence of dyes on the persistence length, the total contour length, and the morphology of individual DNA molecules. We tested three widely used dyes known to differently bind DNA molecules, namely PicoGreen, Dapi, and DRAQ5. Based on our measurements, when imaged at typical concentrations (manufacturer suggested concentrations used for cell imaging), PicoGreen dye showed little effect, Dapi dye decreased the DNA persistence length, and DRAQ5 decreased the persistence length and elongated the DNA. When used at high concentrations, all of the dyes induced drastic changes in the DNA morphology. Our study clearly shows that DNA-binding dyes, irrespective of their DNA binding mechanisms, strongly influence the physical properties of DNA. These changes are strongly dose and dye type dependent and therefore should be taken into consideration when conducting experiments with DNA. ■ INTRODUCTIONFluorescence microscopy is a widely used technique in cell biology, biochemistry, and medicine among other fields of scientific research. The need to label and visualize DNA and chromatin is gaining importance in different fields of biological research, with experiments ranging from measurements on isolated DNA 1−6,11−13 to experiments in fixed cells and live cells. [7][8][9][10]14 There is a large variety of DNA labeling dyes: some binding to the minor/major grooves of double-stranded DNA, others intercalating into DNA, and some doing both. 1,8−20 The influence of DNA labeling dyes on the DNA structure and its properties is still a topic of debate, since no clear data exist on how strongly they influence physical properties. 11−19 Nevertheless, several works revealed that parameters such as the contour length and the persistence length of bare DNA can be significantly changed by fluorescent dyes upon binding. 11−20On the example of YOYO-1 dyes, Gunther et al. showed that the intercalation of the dye in double-stranded DNA modified its mechanical and structural properties. 11 Different studies showed that upon binding of YOYO dye the contour length of the DNA increased roughly linearly, reaching 30−40% elongation compared to bare DNA. In experiments performed with optical tweezers Sischka et al. compared different DNA binding agents with different binding modes (such as ethidium bromide, YO-1, distamycin-A, and YOYO), revealing changes in the mechanical response of DNA pulling as the function of the corresponding dye binding mechanisms. 16 Wojcik et al. showed that DRAQ5 dye detached H1 and H2B histones in a concentration dependent manner in live cells. 14 Hig...

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Section: ■ Conclusionmentioning

confidence: 78%

“…Interestingly, this dye is often used for live cell imaging. 8,9,14,37 Its excitation maximum is at 646 nm and emission spectra at 697 nm.…”

Section: ■ Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Influence of DNA Binding Dyes on Bare DNA Structure Studied with Atomic Force Microscopy

et al. 2015

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“…The inability to specifically label the nucleus in cells rich in cytoplasmic RNA makes it difficult or impossible to discern useful pathologic features, such as nuclear spatial density, nuclear-to-cytoplasmic ratio, nuclear pleomorphism, and nucleomegaly, which are important features of neoplasia assessed on H&E pathology. DRAQ5, on the other hand, is a live-cell nuclear stain with excitation and emission properties in the far red to near infrared spectrum (λ ex = 647 nm, λ em = 665–780 nm) that binds DNA stoichiometrically and exclusively, and is commonly used as a marker for DNA content in flow cytometry [24, 25]. The nuclear specificity of DRAQ5 suggests its potential use as a hematoxylin replacement in a fluorescent H&E analog.…”

Section: Introductionmentioning

confidence: 99%

DRAQ5 and Eosin (‘D&E’) as an Analog to Hematoxylin and Eosin for Rapid Fluorescence Histology of Fresh Tissues

et al. 2016

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Real-time on-site histopathology review of biopsy tissues at the point-of-procedure has great potential for significant clinical value and improved patient care. For instance, on-site review can aid in rapid screening of diagnostic biopsies to reduce false-negative results, or in quantitative assessment of biospecimen quality to increase the efficacy of downstream laboratory and histopathology analysis. However, the only currently available rapid pathology method, frozen section analysis (FSA), is too time- and labor-intensive for use in screening large quantities of biopsy tissues and is too destructive for maximum tissue conservation in multiple small needle core biopsies. In this work we demonstrate the spectrally-compatible combination of the nuclear stain DRAQ5 and the anionic counterstain eosin as a dual-component fluorescent staining analog to hematoxylin and eosin intended for use on fresh, unsectioned tissues. Combined with optical sectioning fluorescence microscopy and pseudo-coloring algorithms, DRAQ5 and eosin (“D&E”) enables very fast, non-destructive psuedohistological imaging of tissues at the point-of-acquisition with minimal tissue handling and processing. D&E was validated against H&E on a one-to-one basis on formalin-fixed paraffin-embedded and frozen section tissues of various human organs using standard epi-fluorescence microscopy, demonstrating high fidelity of the staining mechanism as an H&E analog. The method was then applied to fresh, whole 18G renal needle core biopsies and large needle core prostate biospecimen biopsies using fluorescence structured illumination optical sectioning microscopy. We demonstrate the ability to obtain high-resolution histology-like images of unsectioned, fresh tissues similar to subsequent H&E staining of the tissue. The application of D&E does not interfere with subsequent standard-of-care H&E staining and imaging, preserving the integrity of the tissue for thorough downstream analysis. These results indicate that this dual-stain pseudocoloring method could provide a real-time histology-like image at the time of acquisition and valuable objective tissue analysis for the clinician at the time of service.

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“…All samples were processed within 5 days from extraction at the Fundació n Jiménez Díaz in accordance with current FCI recommendations for processing CSF samples in search of hematological malignancies. 16 An aliquot of the CSF sample received was used for cell count using the fluorescent dye DRAQ5 (Biostatus Limited) for DNA staining 17,18 and Perfect-COUNT microspheres (Cytognos). The remaining sample was equally distributed into 2 different aliquots (tube 1 and tube 2).…”

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confidence: 99%

Role of flow cytometry immunophenotyping in the diagnosis of leptomeningeal carcinomatosis

et al. 2011

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Experimental design: Cerebrospinal fluid (CSF) samples from 78 patients who received a diagnosis of epithelial-cell solid tumors and had clinical data suggestive of leptomeningeal carcinomatosis (LC) were studied. A novel FCI protocol was used to identify cells expressing the epithelial cell antigen EpCAM and their DNA content. Accompanying inflammatory cells were also described. FCI results (positive or negative for malignancy) were compared with those from CSF cytology and with the diagnosis established by the clinicians: patients with LC (n ¼ 49), without LC (n ¼ 26), and undetermined (n ¼ 3).Results: FCI described a wide range of EpCAM-positive cells with a hyperdiploid DNA content in the CSF of patients with LC. Compared with cytology, FCI showed higher sensitivity (75.5 vs 65.3) and negative predictive value (67.6 vs 60.5), and similar specificity (96.1 vs 100) and positive predictive value (97.4 vs 100). Concordance between cytology and FCI was high (Kp ¼ 0.83), although misdiagnosis of LC did not show differences between evaluating the CSF with 1 or 2 techniques (P ¼ .06). Receiveroperator characteristic curve analyses showed that lymphocytes and monocytes had a different distribution between patients with and without LC.Conclusion: FCI seems to be a promising new tool for improving the diagnostic examination of patients with suspicion of LC. Detection of epithelial cells with a higher DNA content is highly specific of LC, but evaluation of the nonepithelial cell compartment of the CSF might also be useful for supporting this diagnosis.Keywords: flow cytometry, immunophenotype, leptomeningeal carcinomatosis. L eptomeningeal carcinomatosis (LC) is a devastating cancer complication developing in at least 5% -10% of patients with solid tumors. Its prognosis is poor, with a median survival of 3-6 months among patients receiving chemotherapy.1 -3 Early diagnosis of LC and treatment initiation could offer the best chance of controlling symptoms and prevent the establishment of irreversible neurologic deficits that impair the patient's quality of life.3 -5 However, LC diagnosis currently remains a challenge. Cytological identification of malignant cells in the cerebrospinal fluid (CSF) remains the gold standard for LC diagnosis, but up to 45% of patients have an initial negative result of cytological examination of the CSF. This sensitivity may increase up to 90% when a high number of lumbar punctures are performed. 6,7 The implementation of imaging techniques and biochemical analysis of the CSF offers useful information for diagnosis; however, both of them should be considered in the right clinical context, because they lack specificity and their sensitivity is low. 1,3 It is therefore imperative to improve diagnostic tools.Multiparametric flow cytometry immunophenotyping (FCI) is an established and necessary laboratory instrument for diagnosis and follow-up of a wide range of hematological malignancies. In turn, FCI is not routinely used for the study of nonhematological tumors. Today, the number of monoclona...

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DRAQ5 Labeling of Nuclear DNA in Live and Fixed Cells

Cited by 36 publications

References 26 publications

Influence of DNA Binding Dyes on Bare DNA Structure Studied with Atomic Force Microscopy

Influence of DNA Binding Dyes on Bare DNA Structure Studied with Atomic Force Microscopy

DRAQ5 and Eosin (‘D&E’) as an Analog to Hematoxylin and Eosin for Rapid Fluorescence Histology of Fresh Tissues

Role of flow cytometry immunophenotyping in the diagnosis of leptomeningeal carcinomatosis

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