Andor luca s

After treatment of cells, the medium was changed and cells were cultivated until the next day to allow for micronucleus formation. On the next day, cells were placed in the plate chamber of a Nikon Ti–S fluorescence microscope equipped with a motorized table and an incubation envelope (Okolab) to maintain temperature, CO₂ and humidity. The system was controlled by the software NIS-Elements Advanced Research version 5.10.01 (Nikon). The positions of the chosen micronucleated cells were saved with the software. Images were taken every 10 min for 96 h, covering up to four to five mitoses (cell cycle durations). The originally selected cells are denominated F0, whereas the following generations are denominated F1–F5. Subsequently, sequences for each position were generated. A 40 × 0.75 NA objective (Nikon) and an Andor Luca S (Andor Technology) camera with ND 64 and exposure time of 100 ms were used without binning. Each experiment was repeated five times. Two types of untreated controls were included, one subtype was micronucleated cells and the other subtype was micronucleus-free cells.

All imaging experiments were conducted on glass bottom plates (Cellvis). After treatment of HeLa-H2B-GFP cells, medium was changed and treated cells were cultivated for 24 h to allow for micronucleus induction. Only after radiation, cells were trypsinised from petri dishes and transferred to a glass bottom plate. On the following day, the cell plate was mounted in the live-imaging chamber of a Nikon Ti-S fluorescence microscope equipped with a motorized table and an incubation envelope (Okolab). Temperature, CO₂ and humidity were maintained. The software NIS-Elements Advanced Research version 5.10.01 (Nikon) was used to control the system. Only cells with micronuclei were selected and positions saved for tracking these cells. Every 10 min images were taken for 96 h. Selected micronucleated cells were termed F0, the following generations were termed F1–F5. Images were taken with an Andor Luca S (Andor Technology) camera with ND 64 and exposure time of 100 ms without binning. 40 × 0.75 NA objective (Nikon) was used. Similar experiments with HeLa-H2B-GFP-Lamin B1-dsRed and HeLa-H2B-GFP-LC3B-dsRed cells were conducted after treatment with etoposide or tBHP at ND 8, 300-ms exposure time and 2 × 2-Binning using z-stacks with 5 levels in 2.5 µm steps around focal plane.