Prostate cancer cell lines DU145 and PC3 were from Cancer Research UK Laboratories (Clare Hall, Hertfordshire, UK), and 22Rv1 and LNCaP from Professor Sir Walter Bodmer (Dept. of Oncology, University of Oxford, UK). Cell line identity was validated by STR genotyping. DLD-1 colorectal cancer cells expressing (BRCA2+/-) or lacking (BRCA2-/-) BRCA2 were from Dr. Scott Kern (Laboratory of Cellular and Molecular Biology, NIH, Baltimore, MA, USA). We used IGF-1R inhibitor AZ12253801 (AstraZeneca, Alderley Park, UK), described in11 , human R3 IGF-1 (Sigma-Aldrich, USA), CDK1 inhibitor RO-3306 and RAD51 inhibitors RL-1 and BO2 (Calbiochem, Merck Millipore, Watford, UK). Cells underwent Cesium-137 irradiation in an IBL 637 irradiator (CIS Bio International, Bagnols/Ceze, France). Gene silencing and western blotting were performed as11 using siRNAs and antibodies listed in Supplementary information. Cell viability was quantified by CellTiter-Glo (CTG) Luminescent assay (Promega, USA), and clonogenic survival, cell cycle distribution and immunofluorescent detection of γH2AX and RAD51 foci as in11 and Supplementary information.
Ibl 637 irradiator
Manufactured by PerkinElmer
Sourced in France
The IBL-637 irradiator is a laboratory instrument designed for research applications. It provides a controlled source of ionizing radiation for sample preparation and analysis. The device operates using a radioactive source to generate a uniform radiation field within its shielded chamber.
Automatically generated - may contain errors
4 protocols using ibl 637 irradiator
1
Prostate and Colorectal Cancer Cell Lines
2
Ionizing Radiation in Epidermal Regeneration
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Exposition of skin substitutes to ionizing radiation was performed at the initial step of epidermis regeneration by keratinocyte precursor cells. Accordingly, samples were irradiated 6 h after keratinocyte seeding onto dermal reconstructs, a time sufficient for their recovery from trypsinization and attachment to their new environment. A 137Cs source was used (γ rays, IBL637 irradiator, Cis-Bio international, Saclay, France). Low dose irradiations (50 mGy) were performed at the dose rate of 50 mGy/min, and irradiations at higher dose (2 Gy) at the dose rate of 850 mGy/min. Control samples were sham-irradiated (same processing except γ ray delivery). The control, 50 mGy and 2 Gy cohorts comprise respectively 14, 14 and 13 skin substitutes.
3
Germ Corpse Counting in C. elegans
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Synchronized young adult animals (AD2-3) grown on RNAi bacteria at 25º were stained with 75 µg/ml acridine orange (AO)(Sigma) for 1 hour, and transferred to fresh NGM agar plates for 3 hours to clear excess AO from their intestines. AO positive germ corpses present in the posterior gonads were counted using a fluorescence dissection stereomicroscope (Leica Microsystems), for each indicated condition 40 animals were counted. Irradiated animals were analyzed 24-36 hours post irradiation with the indicated doses (IBL-637 irradiator, CIS Biointernational).
4
Western Blotting and Immunostaining Protocols
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For Western blotting, proteins were transferred to either nitrocellulose or PVDF membranes, probed with the indicated antibodies, and imaged in a Bio-Rad ChemiDoc imaging system. For an overview of all antibodies used in this study, see Table 1.
For (immuno)staining, cells were grown on coverslips, fixed in 2% formaldehyde, permeabilized with 0.1% Triton-X100 and incubated for 15 minutes with 0.5% BSA and 0.1% glycine solution in PBS. ProteoStat® staining (ENZO, ENZ-51023-KP050) was performed according to the manufacturer's instructions. Primary antibody incubation (see Table 2) was performed overnight at 4°C. After secondary antibody incubation cells were stained with Hoechst (Invitrogen, H1399) and mounted on microscopy slides in Citifluor (Agar Scientific).
Cells were observed using a confocal scanning microscope (Leica), and images were analyzed using ImageJ software. The aggresome signature was defined as cells exhibiting both a curved nucleus and perinuclear presence of ProteoStat® dye. For DNA repair kinetics experiments, cells were irradiated with 2 Gy (IBL-637 irradiator, CIS Biointernational), fixed at the indicated timepoints post-irradiation, and stained for 53BP1. Images were analyzed using ImageJ software.
For (immuno)staining, cells were grown on coverslips, fixed in 2% formaldehyde, permeabilized with 0.1% Triton-X100 and incubated for 15 minutes with 0.5% BSA and 0.1% glycine solution in PBS. ProteoStat® staining (ENZO, ENZ-51023-KP050) was performed according to the manufacturer's instructions. Primary antibody incubation (see Table 2) was performed overnight at 4°C. After secondary antibody incubation cells were stained with Hoechst (Invitrogen, H1399) and mounted on microscopy slides in Citifluor (Agar Scientific).
Cells were observed using a confocal scanning microscope (Leica), and images were analyzed using ImageJ software. The aggresome signature was defined as cells exhibiting both a curved nucleus and perinuclear presence of ProteoStat® dye. For DNA repair kinetics experiments, cells were irradiated with 2 Gy (IBL-637 irradiator, CIS Biointernational), fixed at the indicated timepoints post-irradiation, and stained for 53BP1. Images were analyzed using ImageJ software.
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