Cells were trypsinized, washed with ice-cold PBS, and fixed at a
concentration of 2 × 106 cells/mL in ice-cold 70%
ethanol. For γH2AX analysis, samples were incubated with a mouse
anti-γH2AX-specific antibody (clone JBW301; Millipore) overnight at
4°C followed by incubation with a FITC-conjugated secondary antibody
(Sigma) as previously described (30 (link)). For
quantification of γH2AX positivity, a gate was arbitrarily set on the
control, untreated sample to define a region of positive staining for
γH2AX of approximately 5%. This gate was then overlaid on the
drug/radiation-treated samples. For pHistone H3 analysis, cells were processed
as described above, then incubated with a rabbit anti-pHistone H3 (S10) antibody
(Millipore) as previously described (31 (link)).
Samples for both analyses were stained with propidium iodide to measure total
DNA content and analyzed on a FACScan flow cytometer (Becton Dickinson) with
FlowJo software (Tree Star).
concentration of 2 × 106 cells/mL in ice-cold 70%
ethanol. For γH2AX analysis, samples were incubated with a mouse
anti-γH2AX-specific antibody (clone JBW301; Millipore) overnight at
4°C followed by incubation with a FITC-conjugated secondary antibody
(Sigma) as previously described (30 (link)). For
quantification of γH2AX positivity, a gate was arbitrarily set on the
control, untreated sample to define a region of positive staining for
γH2AX of approximately 5%. This gate was then overlaid on the
drug/radiation-treated samples. For pHistone H3 analysis, cells were processed
as described above, then incubated with a rabbit anti-pHistone H3 (S10) antibody
(Millipore) as previously described (31 (link)).
Samples for both analyses were stained with propidium iodide to measure total
DNA content and analyzed on a FACScan flow cytometer (Becton Dickinson) with
FlowJo software (Tree Star).