To better visualize the mitochondria in the examined cells, we also performed a double staining experiment using—in addition to EB—Rhodamine 123 (R123), which is a well-known potentiometric probe of mitochondria in living cells. The EB fluorescent image and the R123 image are reported, respectively, in Figs. 2(a) and 2(c). Both probes are localized in mitochondria, but their fluorescence distribution pattern is different. While R123 probes the mitochondrial potential [Figs. 2(c) and 2(d)], EB staining shows highly fluorescent nucleoids inside each mitochondrion [Figs. 2(a) and 2(b). When SHSY-5Y cells were induced to differentiate by treatment with retinoic acid (RA) (10 μM for 72 h), they underwent a morphological change into a neuronal phenotype, protruding their neuritis into the extracellular space.26 In Fig. 3, using cytochrome oxidase IV (COX) immunofluorescence, we compared the SHSY-5Y cells before and after differentiation. As can be seen, before differentiation the cells displayed an epithelioid morphology [Fig. 3(a)], while after differentiation [Fig. 3(b)] they acquired an elongated morphology with the formation of long neurites, responsible for a reduced adherence to the substrate. Moreover, differentiated cells were characterized by a smaller cytoplasm and by a reduced number of mitochondria [Fig. 3(b)] compared to the undifferentiated SHSY-5Y tumor cells [Fig. 3(a)]. Similar images were obtained by EB staining, both for tumor [Figs. 4(a) and 4(b)] and differentiated cells [Figs. 4(c), 4(d), and 5]. Interestingly, in the differentiated cells the overall EB fluorescence was found to be appreciably reduced after 72 h of RA treatment [Fig. 4(c)], even though at this stage only a fraction of the cell population assumed a neuronal morphology. This result indicated that all the differentiating cells displayed a reduction of their EB fluorescence intensity, independently of the phase reached by each cell in its morphological transformation [Figs. 4(c) and 5]. In particular, the EB fluorescent nucleoids in these differentiating cells were found to be strongly reduced in number, compared with undifferentiated tumour cells. The EB fluorescence background along each organelle was also reduced, a result that indicates a decrease in the content of mitochondrial RNA in the differentiated cells.