5 bromo 2 deoxyuridine b5002

The following chemicals were purchased from Sigma-Aldrich: ATM inhibitor KU-60019 (SML1416-5MG), ATR inhibitor AZ-20 (SML1328-5MG), bleomycin (B8416), etoposide (E1383), doxycycline (D3072), cisplatin (P4394), 5-Bromo-2′-deoxyuridine (B5002). Puromycin was purchased from Thermo-Fisher (A1113802), blasticidin S was purchased from Carl Roth (CP14.2).

MEFs were serum-starved for 48 h in the presence of 4-OHT followed by incubation in FBS-containing medium for the indicated times. One hour before collection, cells were incubated in labelling medium containing 10 µM 5-bromo-2’-deoxyuridine (B5002, Sigma) followed by trypsinization, washing with PBS and fixation in 70% ethanol. Following two further washes in PBS, cells were incubated with 2 M HCl for 20 min and probed with anti-BrdU monoclonal antibody (5292, Cell Signaling Technologies) and detected with fluorescein isothiocyanate-conjugated goat anti-mouse IgG2a (Jackson ImmunoResearch Labs). Cells were resuspended in 500 μl propidium Iodide solution (50 mg ml⁻¹; P4864, Sigma) and RNase A (100 µg ml⁻¹; R5503, Sigma) and analyzed using a BD Accuri C6 flow cytometer (BD Bioscience) and FlowJo software.