Microscopic dishes

Fluorescence images were acquired, analyzed, and quantified using an Axiovert 200 M fluorescence microscope (Zeiss, Oberkochen, Germany) or an automated high-content screening microscope Array Scan VTI (Thermo Fisher, Dreieich, Germany) as described [39 (link),93 (link),94 (link)]. We seeded cells in microscopic dishes (35 mm, MatTek, Ashland, MA, USA) or clear-bottom 96-well plates (Greiner, Kremsmünster Austria) and fixed them with 4% PFA (20 min, RT). For immunofluorescence staining, we additionally permeabilized the cells via incubation with Triton-X 100 (0.1%, 10 min, RT). Antibodies were diluted in 10% FBS/PBS and incubated with samples for 1 h at RT. We washed the cells (n = 3) in PBS and then incubated the samples with fluorophore-labeled antibodies for 1 h at RT. Finally, we stained the nuclei by adding Hoechst 33342 (50 ng/mL in PBS) for 30 min at RT. For automated high-content screening, regions of interest were created using the nucleus signal and each sample was acquired in triplicate, imaging at least 5000 events per sample according to [39 (link)].

Fluorescence images were acquired, analyzed, and quantified using Axiovert 200 M fluorescence microscope (Zeiss) or the automated high-content screening microscope Array Scan VTI (Thermo Fisher) as described [1 (link),33 (link),34 (link)]. We seeded cells in microscopic dishes (35 mm, MatTek) or clear-bottom 96-well plates (Greiner) and fixed them with 4% PFA (20 min, RT). For immunofluorescence staining, we additionally permeabilized the cells via incubation with Triton-X 100 (0.1%, 10 min, RT). Antibodies were diluted in 10% FBS/PBS and incubated with samples for 1 h at RT. We washed the cells (n = 3) in (PBS) and then incubated the samples with fluorophore-labeled antibodies for 1 h at RT. Finally, we stained the nuclei by adding Hoechst 33342 (50 ng/mL in PBS) for 30 min at RT. For automated high-content screening, regions of interest were created using the nucleus signal and each sample was acquired in triplicate, imaging at least 5000 events per sample according to [1 (link)].