Protocol detail

Imaging Mitotic Aberrations in Glioblastoma Cells

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U87/H2B-mCherry/Centrin2-EGFP and U87/shp53/H2B-mCherry/Centrin2-EGFP cells were seeded on 4-chamber, glass-bottom CELLview tissue culture dishes (Grenier Bio-One) and allowed to grow for 48–72 hrs. Time-laps videos were captured using a Zeiss Cell Observer SD spinning disk confocal microscope as described (26 ). AZ32 was added 1 hr before irradiation (5 Gy). Images were taken every 7 min beginning 2 hrs after irradiation for a total of 16 hrs. Aberrant mitoses were identified visually by morphological abnormalities in chromatin and/or centrosomes (27 (link)). DNA was visualized by DAPI stain or by expression of a fluorescent histone H2B-mCherry fusion protein. Centrosomes were fluorescently labelled with antibodies against α-tubulin or visualized with an EGFP-Centrin2 fusion protein.

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Karlin J., Allen J., Ahmad S.F., Hughes G., Sheridan V., Odedra R., Farrington P., Cadogan E.B., Riches L.C., Garcia-Trinidad A., Thomason A.G., Patel B., Vincent J., Lau A., Pike K.G., Hunt T.A., Sule A., Valerie N.C., Biddlestone-Thorpe L., Kahn J., Beckta J.M., Mukhopadhyay N., Barlaam B., Degorce S.L., Kettle J., Colclough N., Wilson J., Smith A., Barrett I.P., Zheng L., Zhang T., Wang Y., Chen K., Pass M., Durant S.T, & Valerie K. (2018). Orally bioavailable and blood-brain-barrier penetrating ATM inhibitor (AZ32) radiosensitizes intracranial gliomas in mice. Molecular cancer therapeutics, 17(8), 1637-1647.

Publication 2018

Cell Observer SD

Abnormalities Antibodies Cell Centrosomes Chromatin Confocal microscope Dapi Dishes Egfp protein Histone h2b Irradiation Laps Mcherry fluorescent protein Mitoses Stain Tissue α tubulin

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Variable analysis

independent variables

AZ32 treatment
Irradiation (5 Gy)

dependent variables

Aberrant mitoses
Chromatin and/or centrosome morphological abnormalities

control variables

Cell lines (U87/H2B-mCherry/Centrin2-EGFP and U87/shp53/H2B-mCherry/Centrin2-EGFP)
Time-lapse imaging using Zeiss Cell Observer SD spinning disk confocal microscope
Image acquisition frequency (every 7 min for 16 hrs)
DNA visualization (DAPI stain or H2B-mCherry fusion protein)
Centrosome labeling (α-tubulin antibodies or EGFP-Centrin2 fusion protein)

controls

No positive or negative controls explicitly mentioned.

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