EGFR, syntenin, and flotillin-1 in endosomes of cells expressing ALIXΔPRR. HeLa-MZ cells were transfected for 18 h with ALIXΔPRR-mCherry (A) or mCherry (not shown) as a control; only 30–40% of the cells were transfected to facilitate the compared analysis of transfected versus untransfected cells. After fixation, cells were labeled with antibodies against LAMP1 as well as antibodies against EGFR, syntenin, or flotillin-1 (panels showing CD81 or CD63, GM130, and calnexin are shown in Fig. 9). Samples were then processed for high-throughput automated triple-channel fluorescence microscopy. Cells were segmented using the ALIXΔPRR-mCherry or the mCherry signal to identify transfected and untransfected cells. To quantify the distribution of each marker within LAMP1-positive endolysosomes, a mask of the LAMP1 staining pattern was created by segmentation, and this mask was applied onto the staining of the given marker (EGFR, syntenin, flotillin-1) so that the integrated intensity per cell of the marker present in LAMP1-positive endolysosomes could be quantified in the mask (Fig. 9 B). To illustrate the image analysis process, the presence of each marker in the LAMP1 mask only was visualized on the computer screen (reflecting exactly what was being quantified), and the corresponding image of the mask was captured. The righthand panels show these screen captures illustrating the staining of each marker within LAMP1-endosomes only (LAMP1 mask). Scale bar: 10 µm.