Csu x1 m1n

Cells were placed in a 37 °C humid incubator by controlling the temperature and CO₂ flow using H201-couple with temperature and CO₂ units. Live chromatin imaging was performed using a DMI8 inverted automated microscope (Leica Microsystems) featuring a confocal spinning disk unit (CSU-X1-M1N, Yokogawa). An integrated laser engine (ILE 400, Andor) was used for excitation with a selected wavelength of 647 nm and 140 mW as excitation power. A 100× oil immersion objective (Leica HCX-PL-APO) with a 1.4 NA was chosen for a high-resolution imaging. Fluorescence emission of the SiR-Hoechst was filtered by a single-band bandpass filter (FF01-650/13-25, Semrock, Inc.). Image series of 150 frames (5 fps), with exposure time of 150 ms per frame, were acquired using Metamorph software (Molecular Devices) and detected using sCMOS cameras (ORCA-Flash4.0 V2) and 1 × 1 binning, with sample pixel size of 65 nm. All series were recorded at 37 °C.

1.5-days-old embryos were electroporated with a pNLS-EGFP-L2-PCNA (Leonhardt et al., 2000 (link)) vector, to distinguish the G2/M/G1 phases of the cell cycle, at 0.5 µg/µl. 6 hr later, embryos were dissected, fluorescent neural tubes were transferred to a tissue chopper (Mc Ilwain) and 100 µm thick transverse sections were sliced. Sections were collected in 199 culture medium (GIBCO) and were sorted out under a fluorescence microscope to control tissue integrity and the presence of isolated fluorescent cells along the dorso-ventral axis. Each slice was imbedded into 10 µl of rat type I collagen (Roche; diluted at 80% with 1X MEM (GIBCO), 1X GlutaMax (GIBCO) and neutralizing bicarbonate (GIBCO)). Four neural tube-containing collagen drops (5 µl) were distributed on a 35 mm glass-bottom culture dish (IBIDI). Collagen polymerization was performed at 38°C for 30 min and 1.5 ml of complete culture medium (199 medium, 5% FCS, 1X GlutaMax, Gentamicin 40 µg/ml) was gently added. For time-lapse, images were acquired on an inverted microscope (Leica inverted DMI8) equipped with a heating enclosure (set up at 39°C), a spinning disk confocal head (CSU-X1-M1N, Yokogawa) a SCMOS camera and a 63X oil immersion objective (NA 1,4–0,7). We recorded 40 µm thick z stacks (2 µm z-steps) at 5 min intervals. IMARIS and ImageJ software were used for image processing and data analysis.