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Flow cytometry

YK Yusuke Kojima
YM Yuka Machida
SP Sowmiya Palani
TC Thomas R. Caulfield
ER Evette S. Radisky
SK Scott H. Kaufmann
YM Yuichi J. Machida
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For detection of cell death, cells were stained first with Annexin V-APC (BD Bioscience, 550474) in binding buffer (10 mM HEPES-NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) for 15 min at room temperature, and then with 100 µg ml−1 of PI in binding buffer. For analyses of DNA content, cells were fixed with 70% EtOH and stained with PI solution (50 µg ml−1 PI, 10 µg ml−1 RNase A, 0.05% NP-40). Two diploid clones of HAP1 (WT #2 and WT #3) were used as controls for cell-cycle analyses. Stained cells were analyzed by BD FACS Canto II (BD Biosiences). Percentages of cells in G2/M phase were estimated using ModFit LT (Verity Software House).

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