Figure 2. Colocalization of DNA-loaded complexes and endosomes.

(A) Representative confocal images of Cy3-labeled Lipofectamine/DNA (Lipofectamine) and DOTAP/DOPC/DNA (Control) complexes (‘red’ signal). In both images intracellular vesicles were labeled with the endosomial marker FM4–64 (‘green’ signal). In detail, after 1 h of incubation of cells with lipoplexes, the FM4–63 marker was added following manufacturer’s instructions. After 1 additional hour of incubation, cells were viewed at the confocal microscope. A single laser line (488 nm) was used to excite both fluorophores, whose emission was then recorded in two spectrally-separate channels (550–600 for Cy3 and 650–700 for FM4–64). (B) ImageJ software (NIH Image; http://rsbweb.nih.gov/ij/) was used to measure colocalization through Mander’s overlap coefficient (M1 = colocalized red/total red). Manders overlap coefficient M1 was calculated according to Costes et al.³⁶ As evident, DNA was largely found inside endosomes. No significant differences between Lipofectamine and Control were found. Colocalization measurements were evaluated for not less than 50 complexes for each formulation.