Fig. 4

Endocytosis of NL_c formulation by trout macrophages.

(A) Flow cytometry time-course comparison of the membrane-bound (dark grey bar) versus the endocyted liposomes (light grey bar) after incubation with 750 μg/ml liposome-encapsulated 25 μg/ml poly (I:C) and 12.5 μg/ml LPS at the indicated times. Data represent means ± SD of three independent experiments. (B) Effect of chemical inhibitors on the endocytosis of NL_c (750 μg/ml liposome-encapsulated 25 μg/ml poly (I:C) and 12.5 μg/ml LPS) macrophages uptake. Inhibitors were used at the following concentrations: MβCD at 5 mM, EIPA at 50 μM, sucrose at 150 mM and W at 100 nM. The uptake of cells not treated with inhibitors (NL_c bar) was used as 100% uptake control and non-treated cells were used as control (control bar). Data represent means ± SD of 3 independent experiments. Differences were analyzed using One-way ANOVA followed by Newman-Keuls post-test. *, p<0.05; **, p<0.01. (C) Confocal microscopy images of fluorescent liposomes (NL_c) endocyted by macrophages. Cells incubated 30 min, 1 h and 16 h with NL_c containing DHPE-Fluorescein (green) at a 0.05 molar ratio. Cell membranes were stained with CellMask (red) and nucleus with Hoechst (blue).