Eclipse ti microscope

Aliquots of cells in early log phase were placed on a pad of agar (2% (w/v) agarose in EMMG, 100 μg ml⁻¹ uridine, 100 μg ml⁻¹ leucine) on a microscope slide. A coverslip was placed on top and sealed with VALAP (Vaseline, Lanolin, Paraffin wax in a 1:1:1 mix). Slides were incubated at 25 °C or 35 °C for > 1 hour prior to imaging. A heated microscope stage was used to maintain the temperature during imaging using a spinning disk confocal microscope: PerkinElmer UltraVIEW VoX confocal imaging system with a Yokagawa CSU-X1 disk unit, Hamamatsu ORCA-R2 camera and a Nikon ECLIPSE Ti microscope controlled by PerkinElmer Volocity v 6.1 software. Images were captured at 0.1 fps using a 100 × 1.4NA objective and 2x camera binning, giving an equivalent pixel size of 139 nm.

Cells were seeded in 1 glass bottom well chamber one day before analysis and pre-sensitized with 10 μM bromodeoxyuridine (BrdU, GE Healthcare) for 24 h. Before laser irradiation, cells were incubated with 1 µM THZ1 (1604810-83-4) for 1 h, at 37 °C and 5% CO2. The cells were then maintained under the same conditions using the Temperature Control Chamber (PerkinElmer UltraView VoX), and images were taken with a Nikon Eclipse Ti microscope equipped with a 63x oil objective and equipped with a Perkin Elmer spinning disk. Images were collected every 4 s for 10 min after irradiation. Cellular nuclei were irradiated with a 355 nm UV ablation laser at a power setting of 0.15, a repetition rate of 200 Hz, a pulse energy >60 μJ, pulse length< 4 ns (Rapp OptoElectronic). The intensity of the GFP signal was measured using ImageJ software for at least 20 cells per condition from three biological replicates.