Mplus av software package

Subcultures of patient fibroblasts were seeded at a density of 4000/cm² in DMEM with stable glutamine and 15% of FCS (both Sigma, St. Louis, MO, USA) and DNA damage was elicited by exposure of cells to 6 MV X‐ray photons provided by a linear accelerator (Primus series, Siemens Healthcare, Erlangen, Germany) at a dose rate of 3 Gy/min at assay time 0. Matched cultures from treated and untreated fibroblasts were harvested 48 hr postirradiation using Accutase (PAN Biotech, Aidenbach, Germany). For cell cycle analysis, detached cells were pelleted and resuspended at a density of 10⁶/ml in staining buffer containing 4 µg/ml of 4′,6‐diamidino‐2‐phenylindole (DAPI), 100 mM of Tris pH 7.4, 154 mM of NaCl, 1 mM of CaCl2, 0.5 mM of MgCl2, 0.2% of BSA, 0.1% of NP40 in dH2O (Sigma) for a minimum of 30 minutes at 4°C in the dark (Darzynkiewicz, Huang, & Zhao, 2017). Bivariate flow histograms were recorded on an analytical, triple laser‐equipped flow cytometer (LSRII, Becton Dickinson, Franklin Lakes, NJ, USA) using UV excitation/emission at 355/460 nm and FACSDiva software for data acquisition. The resulting cell cycle distributions reflecting cellular DNA content and cell cycle progression were quantified by means of the MPLUS AV software package to assess G2 phase accumulation (Phoenix Flow Systems, San Diego, CA, USA) (Schindler & Hoehn, 1999).