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C Humbert

Publications and source records attributed to C Humbert.

6 recordsLinked to original sources
PubMed1992

Methods for topographical analysis of intra-nuclear BrdUrd-tagged fluorescence.

The observation of BrdUrd staining in the nuclei of cells from exponentially growing populations reveals different typical replicating patterns. We propose a methodological approach to order and characterize BrdUrd intranuclear distributions. First, visual ordering of the patterns is assessed using a spectral analysis coupled to a k-nearest neighbors clustering technique. Subsequently, nine topographical features are introduced to characterize the spatial distribution of BrdUrd-tagged fluorescence in the nuclei of proliferating cells. These topographical features are based on a structural approach. The localization of fluorescence spots is expressed in terms of the normalized distance from the nuclear border and its standard deviation. These topographical features are simple to calculate and easy to relate to visual experience.

Algorithms↗
PubMed1992

Eukaryotic DNA replication is a topographically ordered process.

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.

Bromodeoxyuridine↗
PubMed1992

Intranuclear co-location of newly replicated DNA and PCNA by simultaneous immunofluorescent labelling and confocal microscopy in MCF-7 cells.

The intranuclear distribution of newly replicated DNA and of the proliferating cell nuclear antigen (PCNA) was mapped by confocal laser scanning microscopy after simultaneous immunofluorescent labelling of incorporated bromodeoxyuridine (BrdUrd) and PCNA. A mild hydrolysis with HCl followed by an enzymic digestion of DNA was used to produce single-stranded DNA required for BrdUrd immunorevelation, since this procedure preserves PCNA antigenicity. Optical sections obtained with a laser scanning microscope clearly showed a similar distribution of PCNA and BrdUrd within the nuclei, thus confirming previous observations on parallel labelled synchronized cultures. The intranuclear distribution of PCNA and BrdUrd varies concomitantly during the S phase of MCF-7 cells.

Autoantigens↗
PubMed1990

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

This paper addresses the problem of detecting weak incorporation of BrdUrd and the related efficiency of the denaturation protocols used to unmask this thymidine analog. Evidence is presented that measuring the distribution of BrdUrd-tagged fluorescence intensities by image cytometry generates a standard deviation threshold that discriminates between positive and negative MRC5 cells in vitro. A comparison of the thresholding by standard deviation (SDT) with the usual thresholding by the nuclear total fluorescence intensity (FIT) demonstrated that SDT has a significantly higher sensitivity (99.4-100%, depending on the denaturation protocols) than FIT (94.7 and 74.3%, respectively), although both tests have a high specificity (93% and 100%, respectively) for detecting S cells. Since detecting the S cells is not only dependent on the test used, but also on the denaturation protocols, a quality index (QI) was derived from the standard deviation and the mean value of the non-specific fluorescence of negative cell population versus BrdUrd fluorescence of positive cell population. The following DNA denaturation protocols have been assessed according to QI: acidic denaturation, thermal denaturation in formamide, and thermal denaturation in distilled water. Each denaturation procedure was preceded or not by incubation in either proteinase K or Triton X-100. The results showed that thermal denaturation in formamide, especially when preceded by proteinase K incubation, revealed the largest difference between negative and positive cells. This work also demonstrated that image cytometry of BrdUrd-labelled cells can be suitable for clinical application because of the high sensitivity provided and the small samples needed.

Bromodeoxyuridine↗