Individual wells in the plate were imaged consecutively, either until the whole plate was imaged or until 10,000 individual cells were detected. Cell detection was performed based on nucleus segmentation from DAPI staining. All considered markers except for CXCR4 are nuclear markers, so their signal intensities were measured in the segmented nucleus area. The cell surface marker CXCR4 was quantified in a circle around the segmented nucleus. For each cell and marker, we used the average intensity within the respective quantification area as final readout. Each batch of lines for staining included the reference line (‘CTRL0214pf-iely’). This reference line was used to determine parameter values for cell size (usually around 30-400) and an approximate intensity threshold for detecting responding cells.
