A DAPI-Based Modified C-banding Technique for a Rapid Achieving High Photographic Contrast of Centromeres on Chromosomes

Gonen, Raphael; Platkov, Max; Gardos, Ziv; Shayir, Sheli; Levitsky, Inna; Weinstein, Marcelo; Manor, Esther

doi:10.1007/s12013-022-01065-5

Cited by 5 publications

(2 citation statements)

References 19 publications

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“…We preferred to use the anti phospho-histone-H3 (SER 10) mAb as it is highly correlated with chromosome condensation, thus achieving high selectivity for metaphase cells. To further enhance selection of mitotic cells, the cells were synchronized with methotrexate [Gonen et al 2022], and after releasing the cells to undergo mitosis, we used colcemid to interrupt mitosis at metaphase. This procedure allowed isolation of metaphase cells using ow cytometry sorting, thereby enriching the sample with metaphase cells.…”

Section: Discussionmentioning

confidence: 99%

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Metaphase Cells Enrichment for Efficient Use in the Dicentric Chromosome Assay

Platkov¹,

Hadad

Burg

et al. 2022

Cell Biochem Biophys

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The dicentric chromosome assay (DCA), is considered the 'gold standard' for radiation biodosimetry. Yet, DCA, as currently implemented, may be impractical for emergency response applications, especially when time is of the essence, owing to its labor-intensive and time-consuming nature. The growth of a primary lymphocyte culture for 48h in-vitro is required for DCA, and manual scoring of dicentric chromosomes (DCs) requires an additional 24-48h, resulting in an overall processing time of 72-96h for dose estimation.In order to improve this timing. we introduce a protocol that will detect the metaphase cells in a population of cells, and then will harvest only those metaphase cells. Our metaphase enrichment approach is based on xed human lymphocytes incubated with monoclonal, anti-phosphorylated H3 histone (ser 10). Antibodies against this histone have been shown to be speci c for mitotic cells. Colcemid is used to arrest the mitotic cells in metaphase. Following that, a ow-cytometric sorting apparatus isolates the mitotic fraction from a large population of cells, in a few minutes. These mitotic cells are then spread onto a slide and treated with our C-Banding procedure [Gonen et al. 2022], to visualize the centromeres with DAPI. This reduces the chemical processing time to approximately 2 hours. This reduces the time required for the DCA and makes it practical for a much wider set of applications, such as emergency response following exposure of a large population to ionizing radiation.

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Section: Discussionmentioning

confidence: 99%

“…Following that, a ow-cytometric sorting apparatus isolates the mitotic fraction from a large population of cells, in a few minutes. These mitotic cells are then spread onto a slide and treated with our C-Banding procedure [Gonen et al 2022], to visualize the centromeres with DAPI. This reduces the chemical processing time to approximately 2 hours.…”

mentioning

confidence: 99%