Plan apochromat 40x objective

A549-Dual cells were grown on four-well chamber slides (Matsunami Glass, SCS-N04) for 24 h before irradiation at a dose of 8 Gy. At 3 h post-irradiation, the cells were fixed with 4% PFA in phosphate-buffered saline (PBS) for 15 min at room temperature, permeabilized with 0.1% Triton X-100 for 5 min, and blocked with 3% BSA in PBS for 30 min at 37 °C. Following overnight incubation at 4 °C with the primary antibodies anti-phospho-H2A.X (Ser139) (CST, #2577) and anti-dsRNA-K1 (CST, 28764), the cells were incubated with a DyLight 488-conjugated donkey anti-mouse IgG secondary antibody (Invitrogen, SA5-10166) for 30 min at 37 °C. The primary and secondary antibodies were diluted 1:100 and 1:250, respectively, in PBS containing 3% BSA. The cells were then mounted with VECTASHIELD mounting medium with DAPI (H1200, Vector Laboratories). Images were obtained with an all-in-one fluorescence microscope (BZ-X800, KEYENCE, Osaka, Japan) equipped with a Plan Apochromat 40x objective (NA0.95, BZ-PA40, KEYENCE, Osaka, Japan). Positive areas and signal intensities were automatically calculated using a hybrid cell count application (BZ-H4C, KEYENCE, Osaka, Japan) in BZ-X Analyzer software (BZ-H4A, KEYENCE, Osaka, Japan).

The following microscopes were used for analyzing the morphology of cell monolayers: Leica DMi1 equipped with a Leica MC170 HD or Leica Flexacam C1 camera (Leica, Wetzlar, Germany); Eclipse 80i (Nikon, Tokyo, Japan).
Fluorescence microscopic images from immunostained cultured cells and human intestinal organoids were obtained with an iMIC digital microscope (Till Photonics, Gräfelfing, Germany; provided by the group of Manfred Frick from the Institute of General Physiology at the University of Ulm) equipped with an oligochrome light source (390/40 nm excitation filter for Hoechst 33342, 482/20 nm excitation filter for phalloidin-FITC, 640/20 nm excitation filter for Alexa Fluor® 633, 563/20 nm excitation filter for Alexa Fluor® 568), a quadband filter for standard ICC/IHC applications and TIRF, and a CCD Clara camera (Andor). Image processing (cropping; enhancement of brightness and/or contrast) was performed with ImageJ, GIMP or with Microsoft PowerPoint. ImageJ was used for the quantification of the mean fluorescence signal intensity of each organoid.
Fluorescence microscopic images from Vero cells stained with LysoTracker Green DND-26 for visualization of acidic compartments were obtained with the BZ-X810 All-in-One fluorescence microscope (Keyence, Neu-Isenburg, Germany), equipped with a Plan Apochromat 40X objective and BZ-X filters for DAPI (OP-87762) and GFP (OP-87763).