Cells were pulse-labeled with the CellTrace™ CFSE Cell Proliferation Kit (Thermo Fisher Scientific, C34554) according to the manufacturer's instructions. Briefly, cells were incubated with 2 µM CFSE dissolved in PBS for 20 min at 37°C and washed twice with culture medium. For flow cytometry, cells were detached using 0.05% Trypsin-EDTA, resuspended at 105 cells per 1 µl in 1× PBS with 0.5% BSA and 2 mM EDTA. Samples were stained with 1 µg/ml DAPI (Sigma, D9542) before analysis on a BD FACS Canto II HTS flow cytometer.
Non-synchronized cells were pulse-labeled with 10 μM EdU (Life Technologies) for 75 min. After detachment with 0.25% Trypsin-EDTA, cells were fixed with 4% PFA for 20 min, washed with PBS plus 2% FBS followed by 20 min permeabilization with PBS and 0.5% Triton X-100. After a PBS plus 2% FBS wash, cells were incubated with PBS containing 100 mM CuSO4, 1 M sodium ascorbate and 0.2 µM azide Alexa Fluor 647 for 10 min in the dark to reveal EdU incorporation. Samples were stained with 1 µg/ml DAPI before analysis on a BD FACS Canto II HTS flow cytometer. FlowJo was used as analysis software. The cycling index was calculated by calculating the proportion of cells in S and G2/M phase relative to cells in the G0 and G1 phase [(G2M+S)/(G0G1)].
Non-synchronized cells were pulse-labeled with 10 μM EdU (Life Technologies) for 75 min. After detachment with 0.25% Trypsin-EDTA, cells were fixed with 4% PFA for 20 min, washed with PBS plus 2% FBS followed by 20 min permeabilization with PBS and 0.5% Triton X-100. After a PBS plus 2% FBS wash, cells were incubated with PBS containing 100 mM CuSO4, 1 M sodium ascorbate and 0.2 µM azide Alexa Fluor 647 for 10 min in the dark to reveal EdU incorporation. Samples were stained with 1 µg/ml DAPI before analysis on a BD FACS Canto II HTS flow cytometer. FlowJo was used as analysis software. The cycling index was calculated by calculating the proportion of cells in S and G2/M phase relative to cells in the G0 and G1 phase [(G2M+S)/(G0G1)].