Figure 7
IFN-DCs efficiently phagocytose RI-treated SW620 cells. (a) Fluorescence images (left panels) of HLA-DR-stained IFN-DCs (green) performing phagocytosis of PKH26-stained cancer cells (red) into NT or RI spaces, as evaluated by CLSM after 48 h from the beginning of migration in the microfluidic device under competition experimental conditions. The right panels show DIC (Differential Interference Contrast) images of the same fields. Images acquired using a UPLANSAPO 20×/0.75NA objective are shown. Zoom 3; scale bar, 15 μm. To avoid crosstalk between different fluorophores, sequential acquisition was performed. Data are representative of three independent experiments. (b) A 3D rendering of image stacks (16 slices of 0.5 μm Z-step size) acquired within the RI tumor space. Zoom 6; scale bar, 5 μm. One IFN-DC extending trans-cellular dendrites toward SW620 cells is depicted in green (top panel) and one IFN-DC establishing contact with two different SW620 cells is depicted in green (bottom panel). (c) A 3D rendering of one IFN-DC (green) engulfing one SW620 cell (red) from the image stacks (16 slices of 0.5 μm Z-step size) acquired within the RI tumor space (left panel). Orthogonal sections of the phagocytic event depicted in the right panel are shown (left panels). Scale bar 5 μm. In (b and c), a 3D rendering was edited by Adobe Photoshop CC software preserving the original parameters of image acquisition (d) Fluorescence image showing IFN-DCs (green) crossing connecting-channels facing RI space upon phagocytosis of PKH26-stained SW620 cells (red) as evaluated by CLSM after 48 h from the beginning of migration in the microfluidic device, under competition experimental conditions. The right panel shows a DIC image of the same field. Images are acquired as in (a). Scale bar, 15 μm. Representative images of two independent experiments are shown.
