5 ethynyl 2 deoxyuridine (edu)

Cells seeded in the 24-well plate were labeled with 10 μmol/L of EdU (Meilun, Dalian, China) for 2 h at 37 ℃. Subsequently, cells were fixed in 4% of paraformaldehyde for 20 min and incubated in phosphate-buffered saline (PBS) containing 0.5% of Triton- X100 for 15 min. After being washed three times with PBS, 150 μL of dying solution was applied per well for 30 min and nuclei staining with Hoechst 33,342 for 10 min away from light. The proliferating cells were labeled with green fluorescence, with a maximum excitation wavelength of 491 nm and a maximum emission wavelength of 518 nm for 488-Azide. The proliferative rate = EdU-positive cells/Hoechst-positive cells.

The Cell Counting Kit-8 (CCK8, Meilunbio, Dalian, China, MA0218) and 5-ethynyl-2′-deoxyuridine (EdU, Meilunbio, MA0425) labeling assay were used to measure cell proliferation based on the manufacturer’s protocols. In brief, for the CCK8 assay, HA and A172 cells were seeded inside the 96-well microplates and cultured overnight. After incubation with MitoQ for 2 h, the cells were radiated using X-rays at doses of 4 Gy or carbon ion at 2 Gy. Then, 10 μL of CCK-8 reagent was added to each well after 24 h and incubated for 2 h. The optical density (OD) was determined at 450 nm through a multi-plate reader (Tecan Infinite M200, Männedorf, Switzerland).
For the EdU labeling assay, HA and A172 cells were seeded into a 35 mm confocal dish and cultured overnight. After treatment using MitoQ for 2 h, the cells were radiated with 4 Gy X-rays or 2 Gy carbon ion. Based on the manufacturer’s instructions, the EdU kit was utilized for assessing cell proliferative ability after irradiating for 24 h. Hoechst 33,342 (nuclear staining) was utilized to counterstain cells. The images were captured with Laser confocal microscopy.