Foetal bovine serum (fbs)

HeLa cells (obtained from the European Collection of Authenticated Cell Cultures (ECACC), Salisbury, UK) and CHO cells were cultured in Dulbecco’s Minimal Eagle Medium (DMEM; Sigma-Aldrich, Dorset, UK) supplemented with 10% Foetal Bovine Serum (GE Healthcare, Buckinghamshire, UK), 2 mM l-glutamine, 50 units penicillin/mL and 50 µg streptomycin/mL (Sigma-Aldrich); this supplemented DMEM is hereafter referred to as “complete DMEM”. CHOpgsA745 cells (a kind gift from Prof. David J Evans, University of Glasgow, Glasgow, UK) were cultured in Ham’s F12 medium (Sigma-Aldrich) supplemented with 10% Foetal Bovine Serum (GE Healthcare, Little Chalfont, UK), 2 mM l-glutamine, 50 units penicillin/mL and 50 ng streptomycin/mL (Sigma-Aldrich). NK92MI, Daudi and Jurkat cells were cultured in Roswell Park Memorial Institute 1640 Media (RPMI; Sigma-Aldrich) supplemented with 10% Foetal Bovine Serum (GE Healthcare), 2 mM l-glutamine, 50 units penicillin/mL and 50 ng streptomycin/mL (Sigma-Aldrich); supplemented RPMI 1640 is hereafter referred to as “complete RPMI”. Adherent cells were detached using trypsin/EDTA (Sigma-Aldrich). All cells were grown at 37 °C in a humidified atmosphere of 5% CO₂.

Human HeLa cells (cervix, adenocarcinoma; a generous gift from Dr. David Staněk, Institute of Molecular Genetics, Prague), HeLa cells stably expressing FUCCI genes (HeLa-Fucci; a generous gift from Dr. Martin Mistrík, Palacký University, Olomouc) and A549 (lung, carcinoma; ATCC) cells were used. The HeLa cells and HeLa-Fucci were cultivated in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% foetal bovine serum (PAA Laboratories), 3.7 g/l of sodium bicarbonate and 50 μg/ml of gentamicin. The A549 cells were cultivated in Ham's Nutrient Mixture F12 (HyClone) medium supplemented with 10% foetal bovine serum (PAA Laboratories). The cells were cultured in the culture flasks or on coverslips (12 mm in diameter) in a Petri dish at 37°C in a humidified atmosphere containing 5% CO₂.
For the labelling of DNA replication, BrdU was added to the culture medium for 30 minutes (if not stated otherwise). The final concentration of BrdU was 10 μM.
The cells were either fixed with 2% formaldehyde for 10 minutes, washed in 1× PBS, permeabilized in 0.2% Triton X-100 for 10 minutes and washed in 1× PBS or cells were fixed with 70% ethanol for at least 1 hr and washed with 150 mM NaCl and 3 mM KCl.

Human HeLa cells (cervix, adenocarcinoma; a generous gift from Dr. David Staněk, Institute of Molecular Genetics, Prague; [11 (link)]) and HCT116 cells (colon, colorectal carcinoma; a generous gift from Doc. Marián Hajdúch, Palacký University, Olomouc; [11 (link)]) were used. The HeLa cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% foetal bovine serum (PAA Laboratories), 3.7 g/l of sodium bicarbonate and 50 μg/ml of gentamicin. HCT116 cells were grown in McCoy’s medium (Sigma Aldrich) supplemented with 10% foetal bovine serum (PAA Laboratories), 2.2 g/l of sodium bicarbonate and 50 μg/ml of gentamicin. The cells were cultured on coverslips (12 mm in diameter) in a Petri dish or in 96 flat bottom well plates (Orange Scientific) at 37°C in a humidified atmosphere containing 5% CO₂.