Detection of S cells and evaluation of DNA denaturation protocols by image cytometry of fluorescent BrdUrd labelling

Humbert, Catherine; Giroud, Françoise; Brugal, G.

doi:10.1002/cyto.990110406

Cited by 19 publications

(14 citation statements)

References 20 publications

(17 reference statements)

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“…The ranking shows a texture evolution rather than five fixed clusters of patterns. Square side: 15 differences between the last nuclei of one group and the first nuclei of the following group are barely discernable.…”

Section: Classification Of the Brdurd Labelled Nucleimentioning

confidence: 97%

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Eukaryotic DNA replication is a topographically ordered process

Humbert

Usson

1992

Cytometry

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This paper describes the relationship between the BrdUrd replicating pattern of a cell and its localization within the S phase by means of topographical features and DNA content measurement. The present study follows an objective ranking of the BrdUrd patterns obtained from a spectral analysis of the BrdUrd images. The pattern ranking was consistent with the DNA content increase throughout the S phase. Five texture groups were arbitrarily set up for the purpose of multivariate analysis. Nine topographical parameters were computed for each BrdUrd-labelled nucleus. The descriptive quality of these parameters was assessed by means of factorial discriminant analysis. These parameters made it possible to characterize objectively the known pattern distributions of replication sites qualitatively described in the literature. o 1992 Wiley-Liss, Inc. Key terms: Anti-BrdUrd monoclonal antibodies, fluorescence, topographical parameters, image analysisThere is a body of evidence to support the hypothesis that eukaryotic DNA replication occurs as a nonrandom process in a reproducible temporal order (see 35,40 for review); the best known example is the late replication of the inactive X chromosome of mammalian female (5,211.The mechanisms responsible for this fixed replication sequence are not known, but factors such as chromatin condensation, DNA functional activity, or intranuclear arrangement may be related to the S phase ordering on the basis of many observations. It is generally conceded that euchromatin replicates early and heterochromatin replicates late. Chromosomal band analysis reveals that early DNA synthesis corresponds to the Giemsa R-band, while late DNA synthesis corresponds to the G-band (14,21). Holmquist suggested that late replication, which gradually appears during developmental process of embryonic cells along with facultative heterochromatinization, may actively determine gene repression (13).The DNA synthesized in late S might be less important for cell survival than that synthesized in early S. This hypothesis is supported by the data reviewed by Laird et al. (26) indicating that fragile sites in chromosomes of humans, Drosophila, and Microtus represent regions where DNA replicates late. Furthermore, a number of potentially active genes has been shown to replicate early (12). The relationship between gene activity, intra-nuclear arrangement, and replication timing remains unclear, but alteration of genes, as in translocation, is accompanied by changes in their replication time sequence (21). Iqbal et al. explored the question of "the relationship between the temporal replication of a proto-oncogene and its genomic organization" (18). The existence of a relationship between gene location, involving the nuclear matrix arrangement, and DNA replication has been the subject of a number of biochemical studies (8,19,33,41). However, no definitive answer is available yet, due to the variety of nuclear matrix isolation procedures used (8,381.To learn more of the DNA replication process as a function of gene ac...

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Section: Classification Of the Brdurd Labelled Nucleimentioning

confidence: 97%

“…This test was applied to the pattern rank number of nuclei versus their corresponding FI-PI values; 66 nuclei were quantitatively analyzed: 13 nuclei for the first group; 15 nuclei for the second group; 13 nuclei for the third group; 13 nuclei for the fourth, and 12 nuclei for the fifth group.…”

Section: Fluorescence Image Analysismentioning

confidence: 99%

Eukaryotic DNA replication is a topographically ordered process

Humbert

Usson

1992

Cytometry

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“…Indeed, it was not necessary to control the filter set holder or the different settings of the camera automatically ( 5 ) . That would be needed if acquisition was performed field by field, which is the usual procedure (5)(6)(7)(8)(9)(12)(13)(14), rather than condition by condition. However, the slide must be scanned as many times as there are microscopic conditions.…”

Section: Brdurdfi'otal Dna Staining Scatterplotmentioning

confidence: 99%

“…Visual control makes it possible to check all image processing steps and, if necessary, to interact with processing. Fluorescence image analysis is used, in particular, for immunophenotype analysis (5), for intensity evaluation (12)(13)(14), for localization of in situ hybridization signals ( 7 ) , for DNA quantitation (1,6,8,9,15,20), and for living cell analysis (18).…”

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“…These were designed to identify and analyze one cell type in a heterogeneous cell sample under various microscopic conditions, thus making it possible to quantify in a selected cell population both total DNA content using propidium iodide (PI) stain and S-phase fraction using the bromodeoxyuridine (BrdUrd) incorporation technique. The main difference with other methods is that cell images are not all acquired and stored on a disk before analysis (7,8,(12)(13)(14), and image analysis runs condition by condition rather than cell by cell (5)(6)(7)(8)(9)(12)(13)(14). Cells are relocated and measured as many times as necessary.…”

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Methods for cell proliferation analysis by fluorescent image cytometry

et al. 1995

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Methods were developed for multimodal microscopic image analysis in order to identify and analyze one cell type under various microscopic conditions. Our purpose was to quantify both total DNA content using propidium iodide (PI) stain and S-phase fraction using the bromodeoxyuridine (BrdUrd) incorporation technique in cell population subsets. The model chosen was plasma cells in bone marrow triply labelled with fluorescein isothiocyanate (FITC) for intracytoplasmic immunoglobulins, with amino-methylcoumarin-acetate

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