Automated High-Throughput Immunofluorescence Quantification

The images of immunofluorescence were acquired using the automated high throughput fluorescent microscope IN Cell Analyzer 2000 with a Å~20 objective. The fluorophores in wavelength settings were used to generate the IF images (‘DAPI’ for DAPI, ‘FITC’ for AlexaFluor 488 FITC, and ‘Cy5′ for AlexaFluor647). Multiple fields per well were acquired to include a minimum of 100 cells per sample well.
High content analysis (HCA) of the images were processed using the INCell Investigator v.2.7.3 software as described previously (Borghesan et al., 2019 (link), Rapisarda et al., 2017 (link)). DAPI was used as a nuclear mask and a top-hat method allowed the segmentation of cells. To detect cytoplasmic staining in cultured cells, a collar of 7–9 nm around DAPI was applied. Nuclear staining in the reference wavelength, that is all the other wavelengths apart from DAPI, was quantified as an average of pixel intensity (gray scale) within the specified nuclear area. Cytoplasmic IF staining was quantified as a coefficient of variance of the pixel intensities within the collar area in the reference wavelength. In samples of cultured cells, a threshold for positive cells was assigned above the average intensity of unstained or negative control samples.