Alexa 647 conjugated donkey anti mouse ig

Detection of chromatin-bound RPA in S-phase cells followed recently published protocols with the following modifications: 2 × 10⁶ cells were treated with DMSO or 1 μg/ml camptothecin (CPT) for 24 hours. Cells were subsequently washed once with cold PBS, then incubated in cold CSK buffer + 1% Triton-X for 5 min. Cells were then washed with PBS and fixed in 0.5% PFA for 15 min. Cells were subsequently washed twice in BD Perm/Wash buffer (Becton-Dickinson: BD554723) and stained with RPA2/RPA32 antibody (Abcam: ab2175, 1:50) overnight. Cells were then washed with PBS and incubated in secondary antibody (Abcam: ab150113, 1/50 or Alexa 647 conjugated donkey anti-mouse Ig, Invitrogen: A31571, 1/50) for 1 h. Cells were then washed and stained with propidium iodide in the presence of RNAse A overnight. The percentage of S-phase cells as identified by PI staining was quantified by flow cytometry. The gate for RPA-positive cells was established using a negative control sample that was stained with mouse IgG following extraction. RPA-positive cells following DMSO and CPT treatment were subsequently quantified by flow cytometry. A minimum of 10000 cells were counted per biological replicate.