Spinning disk tirf frap

To estimate the proportion of cells that replicate after irradiation in the lung slices, we used a Click-IT chemistry protocol to monitor cell proliferation using 5-ethynyl-2′-deoxyuridine (EdU) incorporation (BCK-EdUPro-FC647). Irradiated slices were incubated in 500 µL of culture medium containing 10 µM EdU. After the desired incubation time (i.e., 24, 48, or 72 h), they were treated according to the manufacturer’s instructions. Then, organotypic lung slices were washed in PBS and incubated with a nuclear dye (e.g., DAPI). For imaging, organotypic lung slices were transferred into a glass support adapted for microscopy (µ-Slide 4 Well Glass Bottom, Ibidi) and imaged on an inverted Nikon Spinning disk TIRF-FRAP using a 10× objective. Per slice, 3 to 5 fields of view were acquired, each containing 50 stacks spaced by 3 µm. The proportion of EdU+ cells was quantified with a semi-automatic method combining 3D reconstruction and segmentation of the nuclei using IMARIS software Bitplane.

To monitor the cell death induced after irradiation in the lung slices, we stained the organotypic lung slices with Hoechst and Ethidium-1 homodimer 24 h after exposure to doses ranging from 3 to 9 Gy. Organotypic lung slices were incubated in 500 µL of culture medium containing 2 µM Ethidium-1 homodimer. Then, organotypic lung slices were washed in PBS and incubated with the Hoechst nuclear dye for 2 h. For imaging, organotypic lung slices were transferred into a glass support adapted for microscopy (µ-Slide 4 Well Glass Bottom, Ibidi, Gräfelfing, Germany) and imaged on an inverted Nikon Spinning disk TIRF-FRAP using a 20× objective. Per slice, 3 to 5 fields of view were acquired, each containing 20 stacks spaced by 3 µm. The proportion of EdU+ cells was quantified with a semi-automatic method combining 3D reconstruction and segmentation of the nuclei using IMARIS software version 9.3.1 (Bitplane, Belfast, UK).