Figure 2. Calcein does not penetrate cellular membranes but is taken up by fluid-phase endocytosis.

HeLa cells (2.5×10⁴/cm²), grown on glass cover-slips for 24 h, were exposed for 2 h to 5 μM calcein under otherwise standard culture conditions and then transferred to fresh culture medium without calcein and followed by confocal laser scanning microscopy for the next 24 h. Before each observation, lysosomes were labelled for 60 min with Lysotracker Red (final concentration 50 nM). (A) Calcein is mainly localized in early and late endosomes in the periphery of the cells, while some has reached a few centrally placed lysosomes (see yellow lysosome-sized granules in the merged picture). When the calcein-containing medium was replaced with normal medium for (B) 1 h, (C) 6 h, (D) 12 h or (E) 24 h, calcein entered lysosomes in a time-dependent way. However, even after 24 h there was no cytosolic staining. The results clearly demonstrate that the hydrophilic alcohol calcein does not pass plasma or lysosomal membranes. Note that even after 24 h there are several, mainly centrally located lysosomes that show only red fluorescence in the merged picture, indicating that the formation rate of new lysosomes (autophagolysosomes; compare with Figure 6) is substantial, taking into consideration the active fusion/fission processes within the lysosomal compartment that will introduce calcein into newly formed lysosomes.