Figure 3.

Alexa Fluor 488 as a reporter for endo/lysosomal escape in live cell imaging. The report of the naturally occurring phenomenon of the endo/lysosomal escape enhancement of saporin (in this case ^Alexasaporin) was assayed by constant measurement of fluorescence. ECV-304 cells were incubated with 1000 nM ^Alexasaporin at 37 °C for 3 h. Afterwards, most of the internalized toxin accumulated in intracellular vesicles. ^Alexasaporin is visualized in green fluorescence, cell membranes in magenta and cell nuclei in cyan. The addition of 10 µg/mL SA1641 (non-cytotoxic concentration) was defined as t = 0 s. Green colouring of the cells reflects the release of ^Alexasaporin (from t = 1400 s to t = 3500 s). To finish the experiment, SA1641 was added (t = 3640 s) to a final concentration of 80 µg/mL (cytotoxic concentration), which results in total cell membrane disruption (from t = 3640 s to t = 3700 s).