, For the irradiation-injury studies, organoids (in vitro XRT) received

, Mice were euthanized 4 h after treatment. Imaging Detection. For confocal laser-scanning microscopy, the organoids or the tissue was fixed and embedded in agarose or in OCT compound 4583 (Sakura) as already described (37). Sections of 150 ?m (agarose) or 10 ?m (OCT) were obtained by using an HM650V vibratome (Thermo Fisher) or a CM3050S cryostat (Leica), respectively. For antigen retrieval, slides were steamed for 20 min in 10 mM citrate buffer (pH 6) and cooled for 30 min

, Cell Signaling Technology

, Asp175), chicken anti-GFP, issue.1, p.500

, :200; Abgent; AP1802a), rabbit anti-p65 (1:100; Abcam; 7970), mouse anti-Olfm4 (1:200, Abcam; ab13970), rabbit anti-LC3B

, Thermo Fisher), and corresponding secondary antibodies (Alexa Fluor 488, 568, or 647; Thermo Fisher) were used. DNA was stained by DAPI (1 ?g/mL; Thermo Fisher). The sections were mounted with ProLong Gold Antifade Reagent (Thermo Fisher). Images were acquired using an IX-81 (Olympus) or Opterra swept-field confocal microscope (Bruker) equipped with thermic chamber and CO 2 control, p.568

, Flow Cytometric Analysis and Sorting above; the dye was added after dissociation) or live imaging. For total ROS, CellROX Deep Red (2.5 ?M, 30 min at 37°C; Thermo Fisher) and ROSstar 550 (2.5 ?M, 30 min at 37°C; Li-Cor) were used. For mtROS, MitoSOX Red (5 ?M for 30 min at 37°C, Measurement of Intracellular ROS. Total intracellular ROS and mitochondrial ROS were measured by flow cytometry (cf

, Mitochondrial Superoxide indicator; Molecular Probes) was used. For live imaging, DNA was stained with Hoechst, vol.33342

, Thermo Fisher) and cells were washed with DMEM FluoroBrite (Gibco). Resveratrol (40 ?M

, Sigma) was used as an antioxidant agent and directly added to the culture media just before irradiation and during subsequent culture

A. and M. Analyses, Live organoids were incubated for 30 min at 37°C in the dark with 50 nM MitoTracker Deep Red and/or 50 nM LysoTracker (both from Thermo Fisher). DNA staining and washes were performed as described above. Bafilomycin A1 (Sigma) was added to the culture media for organoid-formation efficiency tests at a concentration of 100 nM as determined by a dose-response study

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