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. 2013 Nov 14;134(6):1495–1503. doi: 10.1002/ijc.28454

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© 2013 UICC

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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In the absence of XPF, oxaliplatin treatment causes persistence of double-strand breaks in mammalian cells. (a) CHO cells (10,000) were treated with 10 µM oxaliplatin (OX) for 2 hr in a 96-well plate and fixed in 4% paraformaldehyde at various time points, blocked with 0.1% Triton X-100 and 1% bovine serum albumin (BSA) in PBS before being stained for γH2AX (b) and 53BP1 (c) foci (1:2000 for γH2AX and 1:1000 for 53BP1 in 1% BSA) overnight. A 1:500 dilution of Alexa fluor 488-labeled secondary antibody was diluted in 1% BSA in PBS and incubated for 1 hr at room temperature, and then cells were stained with a 1:2000 dilution of 1 mg/ml DAPI (Sigma Aldrich) for 10 min at RT before being analyzed on IN Cell Analyser 1000 (GE Life Sciences), counting cells with more than 8 foci per nucleus. Data (b,c) are plotted versus time for WT DMSO (), WT 10 µM oxaliplatin (), XPF DMSO () and XPF 10 µM oxaliplatin (). (n = 3, error bars = standard deviation).

Inline graphic — In the absence of XPF, oxaliplatin treatment causes persistence of double-strand breaks in mammalian cells. (a) CHO cells (10,000) were treated with 10 µM oxaliplatin (OX) for 2 hr in a 96-well plate and fixed in 4% paraformaldehyde at various time points, blocked with 0.1% Triton X-100 and 1% bovine serum albumin (BSA) in PBS before being stained for γH2AX (b) and 53BP1 (c) foci (1:2000 for γH2AX and 1:1000 for 53BP1 in 1% BSA) overnight. A 1:500 dilution of Alexa fluor 488-labeled secondary antibody was diluted in 1% BSA in PBS and incubated for 1 hr at room temperature, and then cells were stained with a 1:2000 dilution of 1 mg/ml DAPI (Sigma Aldrich) for 10 min at RT before being analyzed on IN Cell Analyser 1000 (GE Life Sciences), counting cells with more than 8 foci per nucleus. Data (b,c) are plotted versus time for WT DMSO (), WT 10 µM oxaliplatin (), XPF DMSO () and XPF 10 µM oxaliplatin (). (n = 3, error bars = standard deviation).