Immunostaining and fluorescence microscopy

Approximately 12,000 cells were plated in each well of a 24-well glass bottom plate (MatTek, MA), corresponding to approximately 20% surface coverage to ensure single-cell dispersion. After 16 hours of incubation, cells were fixed with 3.7% paraformaldehyde for 12 min at room temperature. Cells were then permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) for 10 min; nonspecific binding was blocked with PBS supplemented with 1% albumin from bovine serum for 40 min. Nuclear DNA was stained with Hoechst 33342 (Sigma-Aldrich) at 1:50 dilution; F-actin was stained with phalloidin Alexa Fluor 488 (Invitrogen) at a 1:40 dilution. Fluorescently labeled cell samples were visualized with a Nikon digital sight DS-Qi1MC camera mounted on a Nikon TE300 epifluorescence microscope (Nikon Melville, NY) and equipped with a motorized stage and motorized excitation and emission filters (Prior Scientific, Rockland, MA) controlled by NIS-Elements (Nikon). For each sample, 81 (9-by-9 square grid) fields of view from a low-magnification lens (10× Plan Fluor lens; numerical aperture, 0.3; Nikon) were used, which covered a contiguous area of 6.03 mm by 4.73 mm (28.5 mm²). The fluorescence channels for Hoechst 33342 and Alexa Fluor 488 were recorded to obtain the necessary morphometric information about the nucleus and cellular body of each individual cell within the scanning region.