Influence of Cell Fixation on Chromatin Topography

Kozubek, Stanislav; Lukášová, Emı́lie; Amrichová, Jana; Kozubek, Michal; Lišková, Andrea; Šlotová, Jana

doi:10.1006/abio.2000.4538

Cited by 47 publications

(34 citation statements)

References 15 publications

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“…Samples for fixation in Carnoy's fixative (60% absolute ethanol, 30% chloroform, 10% glacial acetic acid) and methanol-acetic acid (Meth-Ac; 3:1 mixture) were incubated in the fixative for 5 minutes. After the first fixation step, samples for Meth-Ac fixation were washed briefly in fresh fixative and then incubated in more fresh fixative for a further 10 minutes [27].…”

Section: Cell Culture and Fixationmentioning

confidence: 99%

Studies of chemical fixation effects in human cell lines using Raman microspectroscopy

Meade¹,

Clarke²,

et al. 2010

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Section: Cell Culture and Fixationmentioning

confidence: 99%

Studies of chemical fixation effects in human cell lines using Raman microspectroscopy

Meade¹,

Clarke²,

et al. 2010

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“…Such doses are much smaller than those used to fix cells (molar concentrations) and allow survival of the cells which are then able to repair the damage induced by FA Schmid and Speit 2007]. Molar concentrations obviously kill the cells, but preserve the native structure of somatic chromatin without extracting nucleoproteins [Hendzel et al 1997;Kozubek et al 2000]. Hence, the FA induced sperm DNA damage is most likely not due to a change in the condensation status of sperm chromatin that depends on the binding to protamines (see above).…”

Section: Tunel: Principles and Type Of Damage Detectedmentioning

confidence: 99%

Critical Aspects of Detection of Sperm DNA Fragmentation by Tunel/Flow Cytometry

Muratori

Tamburrino

Marchiani

et al. 2010

Systems Biology in Reproductive Medicine

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Despite the many studies documenting an increase of sperm DNA damage in subfertile and infertile subjects, determining whether this parameter is relevant for the clinic is still an open question. Indeed, results of clinical investigations on sperm DNA damage and outcome of ART procedures are often conflicting as many factors affect the predictive power of these tests. The techniques used to reveal such damage is one of these factors. Techniques detecting sperm DNA damage are indeed numerous and heterogeneous. In addition, it is not obvious that they reveal the same type of DNA damage and that their results are equivalent. One of the available methods to detect sperm DNA damage is the TUNEL assay. Since it was developed in the 1990s the TUNEL assay has been widely employed, becoming one of the most popular strategies to investigate the origin, the mechanism, and the clinical meaning of sperm DNA damage. Our group has used TUNEL coupled to flow cytometry to investigate sperm DNA fragmentation for about 10 years. According to our experience, this technique presents some pitfalls and limitations in the accuracy and reproducibility of the measures of sperm DNA fragmentation. In this review, we discuss several technical features of TUNEL and report the solutions adopted by our group to overcome some of its limitations.

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“…Many of these dyes are restricted to fixed cell samples and show only roughly the distribution of nuclei in cells and tissues (3). Further, fixation of cells often produces undesired artifacts (4). DNA dyes for live cell fluorescence microscopy should match the criteria of low cytotoxicity and phototoxicity combined with low photobleaching (5).…”

mentioning

confidence: 99%

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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Influence of Cell Fixation on Chromatin Topography

Cited by 47 publications

References 15 publications

Studies of chemical fixation effects in human cell lines using Raman microspectroscopy

Studies of chemical fixation effects in human cell lines using Raman microspectroscopy

Critical Aspects of Detection of Sperm DNA Fragmentation by Tunel/Flow Cytometry

DNA labeling in living cells

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