Metaxpress high content image analysis software

The 4% formaldehyde-fixed cells were washed 3 times with PBS and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) in PBS for 15 minutes. Next, after blocking with 5% BSA in PBS for 1 hour, the plates were incubated overnight at 4°C with primary antibodies diluted in blocking solution (5% BSA in PBS), monoclonal anti-αSMA antibody (Dako, clone 1A4, M085, 1:250 dilution) or anti-p21 antibody (Abcam, clone EPR362, Ab109520, 1:1000 dilution). Then, the plates were washed 3 times in PBS and incubated for 1 hour in the dark with secondary Alexa Fluor 594–conjugated goat IgG (Thermo Fisher Scientific) and Hoechst 33342 (Invitrogen, H3570) at 1:500 and 1:10,000 dilutions in PBS, respectively. After washing 3 times with PBS, the plates were sealed and image analysis was performed using ImageXpress Micro system (Molecular Devices). The percentage of αSMA⁺ or p21⁺ cells was calculated using MetaXpress High Content Image Analysis Software (Molecular Devices).

The ImageXpress platform (Molecular Devices LLC, Sunnyvale, CA) is a widefield automated microscope capable of fluorescent, transmitted light, and phase-contrast imaging of fixed- or live-cells. The system optical drive includes a 300-W Xenon lamp as broad-spectrum white light source and 2/3 chip cooled CCD camera and optical train for standard field of view imaging and a transmitted light option with phase contrast. After incubating endothelial cells with CCs-MPs (from 15 minutes to 24h), cells were stained for beta catenin or Ve-Cadherin. 100 pictures were taken per wells (4 wells per condition) and images were analysed with MetaXpress® High-Content Image Analysis Software (Molecular Devices LLC, Sunnyvale, CA).