Figure 7. QD655-COOH cellular uptake mechanisms in dendritic cells.

(A) Mocodazole (10 μg/ml), CytD (10 μg/ml), Wort (100 ng/ml), Ly (10 μg/ml), CRQ (50 μg/ml), CPM (10 μg/ml), Y-27632 (10 μg/ml), SRP (1 μg/ml), U-73122 (10 μg/ml), polyI (10 μg/ml) and FCD (10 μg/ml) were used to investigate the QD655-COOH uptake inhibition at indicated concentration described in the ‘Materials and methods’ section. QD655-COOH at 0.05 nM were incubated with dendritic cells (DCs) associated or without each inhibitor preincubation. The mean QD fluorescence intensity in the cells was evaluated by flow cytometry and normalized to the control (without inhibitors; n = 3/treatment). The intensities for inhibitor treated samples were all statistically lower (p ≤ 0.05) than the control (100%) except for Y-27632. The inhibitors with endocytic inhibitory effects ≥ approximately 50% are indicated by arrows. (B) QD655-COOH uptake inhibition by U-73122 is concentration dependent. QD655-COOH at 0.05 nM were incubated with DCs associated with or without U-73122 at 0.4, 2 and 10 μg/ml. DCs were collected and evaluated by flow cytometry for QD655-COOH fluorescence intensity and normalized to the control (n = 3/treatment). *p < 0.05 indicates significant differences between the U-73122 dosed group and control. (C) QD655-COOH inhibitory effects by some of the endocytic inhibitors (CytD, Noco, Nystatin, CPM, Ly, SRP, U-73122 and FCD) imaged by fluorescence microscopy. The image for each inhibitor was marked with the percentage fluorescence intensity compared with control. Each cell is 8–10 μm in diameter.

CPM: Chlorpromazine; CRQ: Chloroquine; CytD: Cytochalasin D; FCD: Fuicodan; Ly: Ly294002; QD: Quantum dot; SRP: Staurosporine; Wort: Wortmannin.