4.9. Nuclear/Cytoplasmic Ratio Quantification from Immunofluorescence Images

ImageJ software (NIH, Bethesda, MD, USA) was used to create a custom macro to generate an automated tool to quantify the nuclear/cytoplasmic fluorescence ratio of each cell in the images. The first mask, corresponding to the nuclear area, was determined using DAPI fluorescence. The second mask, which was considered to be the cytoplasmic area, is created by homothetic enlargement of the first mask to generate a ring around the nucleus. These two masks were successively applied to quantify the fluorescence of the protein of interest in the nucleus and in the cytoplasmic area. Then, the nuclear/cytoplasmic immunofluorescence ratio was calculated for each cell. The median of the nuclear/cytoplasmic ratio of the control condition was used as a threshold to determine the percentage of cells below and above this threshold in each condition. For each condition, approximately 1000–1500 cells from at least 10 images were analyzed.