Quantifying DNA Damage Response in HeLa Cells

WT HeLa Kyoto cells were seeded into an 8-well Lab-Tek (Thermo Scientific) and grown for 16 h. Then, different concentrations of 4sT (2mM-10mM; Carbosynth) and 50 μM etoposide (Sigma) were added and cells were incubated for 24 h. For immunofluorescence (IF), cells were washed two times with PBS and fixed using 4 % formaldehyde (Sigma) in PBS for 5 min. formaldehyde was quenched using 20 mM TRIS-HCl (Sigma; adjusted to pH 7.5) in PBS for 3 min and washed with PBS. Cells were permeabilized using 0.5 % Triton-X100 (Sigma) in PBS for 10 min. Then, cells were blocked using 2 % BSA in PBS for 30 min at RT, followed by incubation with 1:500 α-phospho-γ-H2A.X (ABCAM ab2893) in 2 % BSA [PBS] for 1.5 h at RT. Then, cells were washed 3x for 5 min using PBS, followed by incubation with 1:1000 α-mouse-AF488 (Molecular Probes A11001) in 2 % BSA [PBS] for 30 min at RT in the dark. Then, cells were washed one time using PBS for 5 min, followed by staining using 1 μg/ml 4,6-diamidino-2-phenylindole (DAPI; Thermo Scientific) for 5 min. Then, cells were washed again for 5 min in PBS. Samples were imaged on a customized Zeiss LSM780 microscope using a 20x, 0.8 NA, Oil DIC Plan-Apochromat objective (Zeiss). Images were analyzed using CellCognitionExplorer 1.0.2^{52 (link)} for segmentation and intensity extraction and Python scripts to visualize the data.