Black 96 well imaging plates

U2OS cells were seeded in black 96-well imaging plates (Greiner bio one, Kremsmünster, Austria). After 24 h, the medium was removed, cells were rinsed with 37 °C pre-heated PBS and fixed in PBS supplemented with 4% paraformaldehyde (PFA, Sigma) and 1 µg/mL Hoechst (Life Technologies, Carlsbad, CA, USA) for 20 min under agitation at room temperature (RT). Cells were rinsed with PBS before addition of a 5% FBS/0.3% Triton X-100/PBS permeabilizing solution for 30 min under agitation at RT. Cells were rinsed with PBS and incubated overnight under agitation at 4 °C with the primary antibody in 1% BSA/PBS. Cells were washed with PBS and incubated for 45 min under agitation at RT with the secondary antibody in 1% BSA/PBS. Finally, cells were washed and processed for image acquisition using a IXM XL BioImager (Molecular Devices, Sunnyvale, CA, USA).