Wild-type (IB10, subclone of E14 129/Ola; Hooper et al., 1987 (link)) and Rad54-/- mouse ES cells (Essers et al., 1997 (link)) were cultured on gelatinized plates (0.1% porcine gelatin (Sigma)). The culture media consisted of 50% Dulbecco's Modified Eagle Medium (DMEM) (high-glucose, ultraglutamine; Lonza), 40% Buffalo rat liver cell-conditioned medium, 10% fetal calf serum (FCS) supplemented with non-essential amino acids, 0.1 mM β-mercaptoethanol, pen/strap, and 1000 U/ml leukemia inhibitory factor (mouse). Cell lines were routinely tested (negative) for mycoplasma contamination.
For imaging, cells were seeded in eight-well glass-bottom dishes (Ibidi), which were coated with 25 µg/ml laminin (Roche) for at least 1 hr. About 30,000 cells in 300 µl medium were plated per well the day before the experiment. Cells treated with IR were irradiated in an Xstrahl RS320 X-Ray generator (195.0 kV and 10.0 mA) at the indicated dose.
For imaging, cells were seeded in eight-well glass-bottom dishes (Ibidi), which were coated with 25 µg/ml laminin (Roche) for at least 1 hr. About 30,000 cells in 300 µl medium were plated per well the day before the experiment. Cells treated with IR were irradiated in an Xstrahl RS320 X-Ray generator (195.0 kV and 10.0 mA) at the indicated dose.