Micronuclei assay by laser scanning cytometry

Smolewski, Piotr; Ruan, Qiongfang; Vellón, Luciano; Darżynkiewicz, Zbigniew

doi:10.1002/1097-0320(20010901)45:1<19::aid-cyto1140>3.0.co;2-g

Cited by 37 publications

(33 citation statements)

References 26 publications

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“…20,32 Cytochalasin B is commonly used to impair cytokinesis, particularly to generate binucleated cells in the "cytokinesis-blocked micronucleus" (CBMN) assays of mutagenicity. [33][34][35] The present data suggest that impairment of cytokinesis by enforcement of cell attachment may provide an alternative approach to generate binucleated cells that may be used in the CBMN assay. An advantage of this approach is the absence of the chemical agent such as cytochalasin B which prevents cytokinesis but may interfere with the tested mutagens affecting their effectiveness in inducing micronuclei.…”

Section: Discussionmentioning

confidence: 75%

Enforced Adhesion of Hematopoietic Cells to Culture Dish Induces Endomitosis and Polyploidy

Huang

Dai²,

Darżynkiewicz³

2005

Cell Cycle

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Cells of epithelial or endothelial lineage when forced to grow in suspension undergo the detachment-induced death termed "anoikis". In the present study we explored the reverse situation, namely the effect of enforcement of hematopoietic lineage cells that are normally maintained in suspension to grow attached. Towards this end murine L1210 or human HL-60 and Jurkat leukemia cells were cultured in slide chambers coated with poly-L-or poly-D-lysine, or with compound 48/80, the polycations attracting them electrostatically. Within minutes after the transfer L1210 cells strongly adhered to bottom surface of the dish and shortly thereafter binuclear, and later on, polynuclear cells become apparent. The frequency of nuclei per cell increased with time and polykaryons with up to 16 nuclei and high DNA ploidy (DI = 16.0) were apparent after 48 h. After 4 days the size (volume) of some polykaryons exceeded by over 340-fold the volume of mononuclear cells. The presence of mitotic figures and abnormal mitotic spindles in adhering polykaryons provided evidence of the impeded cytokinesis that led to endomitosis. Most polykaryons excluded trypan blue, had balanced growth (unchanged protein/DNA ratio compared to monokaryons), and showed no evidence of apoptosis. Individual nuclei within each polykaryon replicated DNA in synchrony. The strong cell attachment and aborted cytokinesis were cell line specific since no significant endomitosis was observed in Jurkat-or HL-60-cells which did not strongly attach to polycation-coated surfaces. Defective cytokinesis and induction of polyploidy by this mechanism, if it occurs in vivo (e.g., mediated by integrins), may lead to aneuploidy and therefore have tumorigenic consequences. The phenomenon offers a novel experimental model for induction of polyploidy and provides an alternative to cytocholasin B to prevent cytokinesis in the mutagenicity cytokinesis-blocked micronucleus (CBMN) assay.

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Section: Discussionmentioning

confidence: 75%

Enforced Adhesion of Hematopoietic Cells to Culture Dish Induces Endomitosis and Polyploidy

Huang

Dai²,

Darżynkiewicz³

2005

Cell Cycle

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“…Therefore, the range of cellular DNA content between 2.2 and 3.8 DI is the most reliable to represent the binucleated cells. Indeed, imaging of cells whose PI fluorescence (DNA content) was within this range confirmed that 80% of these cells were binucleated (Smolewski et al, 2001). The remaining objects were aggregates consisting of two or three cells in close proximity to each other.…”

Section: Imstar Pathfinder Tm Screentox Auto-mnmentioning

confidence: 83%

“…The frequency of MN in binucleated cells detected visually by microscopy at different mitomycin-C concentrations, correlated well with that assessed by LSC. When the frequency of MN in the same specimens was analysed visually by microscopy and compared with that assayed by LSC, in double-blind tests, rather good correlation (r = 0.87) was observed between both assays (Smolewski et al, 2001).…”

Section: Imstar Pathfinder Tm Screentox Auto-mnmentioning

confidence: 96%

“…The greater challenge was to develop methods for use with nucleated cells as described below. DNA and protein of the cells subjected to MN analysis by LSC with fluorochromes of different emission colour is more advantageous than staining DNA alone, because ratiometric analysis of protein/DNA versus DNA content offers better means of MN identification than DNA content alone (Smolewski et al, 2001). A variety of fluorochromes can be used to differentially stain cellular DNA and protein within a given sample.…”

Section: Imstar Pathfinder Tm Screentox Auto-mnmentioning

confidence: 99%

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HUMN project initiative and review of validation, quality control and prospects for further development of automated micronucleus assays using image cytometry systems

Fenech

Kirsch‐Volders

Rössnerová

et al. 2013

International Journal of Hygiene and Environmental Health

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“…The analysis of apoptosis in single cells, cell cultures, and solid tissues was performed successfully by several authors applying a broad variety of assays; analyzed features include Annexin V, loss of mitochondrial transmembrane potential, DNA strand break analysis, alterations in DNA condensation (for a comprehensive overview, see reference 58), and micronucleus formation (59,60).…”

Section: Apoptosismentioning

confidence: 99%