Cell observer

For time-lapse video microscopy, A375P cells expressing Histone H2B-mRFP1 LV-RFP (Crtl) or H2B-GFP LV-GFP (shL3#1 or shL3#2) were synchronized in G1/S after incubation with 2.5 mM thymidine for 20 h and 1.5 × 10⁴ cells were seeded on camera chambers (IBIDI). Mitotic progression was followed by time-lapse microscopy starting 5 h after release in fresh media. Image acquisition was performed using a Cell Observer (Zeiss) equipped with an inverted light and fluorescence microscope and built with an environmental chamber for controlling temperature, humidity, and CO₂. Light and fluorescence images were acquired with Plan-APOCHROMAT (40x AN1.3) objective at 5 min intervals and processed using Axiovision Rel. 4.8 software.

Fluorescence images were recorded in a Cell Observer (Carl Zeiss Microscopy GmbH, Germany) wide-field fluorescence microscope equipped with a mercury arc lamp (Illuminator HXP 120 V) and a rhodamine filterset.
Imaging of arrays with electrons was done in a Crossbeam 540 (Carl Zeiss Microscopy GmbH, Germany), a field emission SEM featuring a double condensor system. Imaging conditions were: 1.5 kV, a beam current of 811 pA, ESB (energy-selective backscatter) detector, grid at 1000 V, 25.2 microseconds dwell time. Using computer-assisted tools in the newly released ZEISS Atlas 5 Array Tomography platform, serial sections were imaged at multiple resolutions: First the whole array (“region”, mosaic of about 70 tiles) was imaged automatically at 1000 nm image pixel size. Then serial sections were recorded automatically at 60 nm pixel size (“section set”). On these section images interesting cells or groups of cells were selected for further high-resolution imaging (“site sets”). These ROIs were automatically imaged over a range of 50–250 serial sections at 5 nm pixel size using a large single (up to 32 k × 32 k pixels) frame for each site.

Cells were labeled with Lysotracker Red DND-99 (Invitrogen) for 2 h at 1000× dilution or 20 μg/mL DQ Green BSA (Invitrogen) overnight in mESC medium before fixing with 4% paraformaldehyde for 20 min at 25 °C. Cells were then stained with Hoechst stain. To induce starvation, cells were cultured in serum free media for 16 h before harvesting. Images were acquired with fluorescence microscopy (Cell Observer, Zeiss).

LX-2 cells were seeded in silicone 2-well inserts (ibidi, Gräfelfing, Germany) in 12-wells at 50,000 cells per silicone well in 100 µL 1% FBS containing DMEM. 2 mL of 1% or 10% FBS containing DMEM were placed outside the silicone insert. After 24 h, silicone inserts were carefully removed, and cells were imaged in 4 positions on a cell observer (Zeiss, Jena, Germany) at 37 °C in 20% O₂ and 5% CO₂ for 24 h. Gap closure was assessed by an in-house script by Juergen Gindlhuber in NIS-Elements v5.20.02 (Nikon, Tokyo, Japan).