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Automation of the cytokinesis-block micronucleus cytome assay by laser scanning cytometry and
its potential application in radiation biodosimetry
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Maxime François1, Kevin Hochstenbach1,2, Wayne Leifert1, and Michael Felix Fenech1
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BioTechniques 57:309-312 (December 2014) doi 10.2144/000114239
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Keywords: Micronucleus assay; automation; laser scanning cytometry
CSIRO Food and Nutrition Flagship, Nutrigenomics and DNA damage, Adelaide, Australia and
Section of Investigative Medicine, Department of Medicine, Imperial College London, London,
United Kingdom
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Supplementary Material and Methods
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Blood collection, lymphocyte isolation and irradiation
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Upon signing informed consent, 8 ml of fresh blood was drawn from 2 healthy male adult volunteers
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into vacutainers (Greiner Bio-One, Frickenhausen, Germany) with lithium heparin anticoagulant.
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Isolated lymphocytes were counted using a cell counter (Coulter Electronics model ZB1, Beckman
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Coulter, Brea, CA, USA), gently resuspended into RPMI 1640 medium (Sigma-Aldrich, Castle Hill,
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Australia) completed with 10% FBS, 2 mM L-glutamine and 1 mM sodium pyruvate solution to a
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concentration of 1 X 106 per ml in a total of 750 μl medium. Cells were transferred as 750 μl aliquots
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into four 24-well plates (Nunc, Roskilde, Denmark) and exposed to 0, 1, 2, or 4 Gy ionizing radiation
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(0.57 Gy/min) using a Faxitron 650 X-ray machine (Faxitron, Tuscon, AZ, USA).
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Cell culture and slide preparation
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Lymphocytes were cultured at 37°C after mitotic stimulation using 30 µg/ml of Phytohaemagglutinin
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(Thermo Fisher, Scoresby, Australia). 44 h after initiation of the lymphocyte cultures, cytochalasin B
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(Sigma-Aldrich, Castle Hill, Australia) was added at a final concentration of 4.5 μg/ml. At 72 h,
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lymphocytes were harvested, counted again and concentration was adjusted to 300,000 cells/ml
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with complete RPMI 1640 medium, as pilot studies showed this to be the optimal concentration for
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preparing slides for LSC analysis. Cells were cytocentrifuged on slides using a Shandon Cytospin 4
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(Thermo Fisher, Scoresby, Australia) at 40 xg before being air-dried and fixed in methanol for 10 min
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at -20°C. Cytoplasm was stained with 0.2% (w/v) Fast Green (Sigma-Aldrich, Castle Hill, Australia) in
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order for the LSC to determine cytoplasm boundaries. Nuclei were counterstained with 4',6-
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diamidino-2-phenylindole (DAPI; 0.4 μg/ml; Sigma-Aldrich, Castle Hill, Australia). Staining procedures
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were performed in the dark at room temperature. Slides were stored at -20°C in a sealed
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microscope slide box with desiccant until LSC and visual scorings were performed.
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Laser Scanning Cytometer and visual scoring
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The automated scoring of MN in lymphocytes was carried out with an iCyte imaging cytometer (iCys
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7.0; Thorlabs, Sterling Virginia, VA, USA). DAPI and Fast Green were excited with 405 nm and 633 nm
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lasers, respectively and their emitted fluorescence were collected with blue and long red photo
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multiplier tubes, respectively. Predefined settings were used for all microscope slides analyzed. The
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settings were empirically determined for the assay and can be modified in any ways by the operator.
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However, once the settings were set for the experiment they were not modified. Therefore, data
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generated for each donor were directly comparable. These settings included the selection of the
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lasers used, voltage of the photomultiplier tubes, threshold values, color of the contours as well as
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minimal area required for a nucleus or a micronucleus to be identified and contoured as such. Once
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images were generated, the software applied a threshold preset manually to identify the signals of
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interest (represented on the images by pixel values). The thresholds were set so that cytoplasm and
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nuclear stains were identified and contoured appropriately. Data for endpoints of the assay (i.e.
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number of cells, circularity values for each nucleus, number of micronuclei) were automatically
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generated by the software and were then exported to excel.
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For each volunteer and IR dose, duplicate cytospots on each slide were then blindly scored using a
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Nikon Eclipse E-600 microscope (Nikon, Sydney, Australia) with 1000× magnification to determine
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the presence or absence of MN in binucleated cells.