Immunofluorescence images were acquired at room temperature using ZEN 2011 software (Zeiss) on an LSM780 microscope (Zeiss) equipped with a GaAsP detector and 25×/0.8 Plan Apochromat or 40×/1.4 enhanced chemiluminescence Plan Apochromat oil differential interference contrast or 40×/1.3 enhanced chemiluminescence Plan-Neofluar oil differential interference contrast objectives (Zeiss).
In vitro live imaging of cultured cells was performed using an UltraView Vox spinning-disk confocal system (PerkinElmer) installed on an AxioObserver Z1 microscope (Zeiss). Images were recorded with an Hamamatsu electron-multiplying charge-coupled device 9100-13 camera using 40×/1.3 enhanced chemiluminescence Plan Neofluar lens (Zeiss). Acquisition of video sequences was done with the Volocity 3D image software (PerkinElmer). Multiple positions were acquired simultaneously. At each position, z stacks were captured every 3 min. Collected images were deconvolved using Huygens deconvolution suite (SVI). Nuclei volumes and cell cycle times were automatically analyzed using Definiens as described previously (Homem et al., 2013 (link), 2014 (link)).
In vitro live imaging of cultured cells was performed using an UltraView Vox spinning-disk confocal system (PerkinElmer) installed on an AxioObserver Z1 microscope (Zeiss). Images were recorded with an Hamamatsu electron-multiplying charge-coupled device 9100-13 camera using 40×/1.3 enhanced chemiluminescence Plan Neofluar lens (Zeiss). Acquisition of video sequences was done with the Volocity 3D image software (PerkinElmer). Multiple positions were acquired simultaneously. At each position, z stacks were captured every 3 min. Collected images were deconvolved using Huygens deconvolution suite (SVI). Nuclei volumes and cell cycle times were automatically analyzed using Definiens as described previously (Homem et al., 2013 (link), 2014 (link)).
