Fig. 6

Flow cytometry and confocal analysis of PLA-NP uptake. a Flow cytometry analysis of A549 cells treated with the endocytic pathways inhibitors followed by 60 or 240 min incubation with 20 µg/mL PLA-NP. ‘P3 Gate’ refers to A549 half population in green fluorescent gate region. ‘Mean Fluorescence’ reflects the analysis of ‘P3 Gate’. Each experimental group corresponds to three independent experiments performed in two replicates. Student t test compared the experimental condition to respective Control groups (*p < 0.05, **p < 0.01). Results are expressed as mean (±SEM). b Representative images of three independent experiments acquired by confocal microscopy of A549 cells treated with lipid rafts and clathrin pits inhibitors followed by 240 min incubation with 20 µg/mL PLA-NP. Green dots refers to PLA-NP, red and blue staining to actin and nuclei, respectively. c Representative images of three independent experiments showing the co-localization of the lipid raft marker CTX-Alexa Fluor 594 and PLA-NP in A549 cells. Green dots refer to PLA-NP, red and blue staining to ganglioside GM1-bound CTX and nuclei, respectively. Cross-hair lines in the Z-stack images evidence green and red fluorescence overlapping, hence co-localization. Scale bar 30 µm