Figure 1.

H-ACs-P2X7 favor the membrane transfer to Mutu dendritic cells

(A). The expression of P2X7R in MutuDCs was evaluated in a functional assay as Ethidium Bromide (20 μg/mL) uptake over time in response to ATP (1 mM). Representative micrographs (every 5 min) are shown; ATP and Triton X-100 addition are indicated. Scale bar 20 µm.

(B) To evaluate the membrane transfer, ACs generated from transfected or parental HEK293 were labeled with the lipophilic stain CM Deep Red , shown as green pseudocolor. Orthogonal projection obtained from Z-stacks denotes the fluorescent membrane.

(C) MutuDCs were challenged with CM-labeled ACs. Transfer of fluorescence was evaluated in a circular region of interest on the outer perimeter of selected cells. The fluorescence over time was plotted for ACs loaded with CM using the ZEN Blue edition 2.6 software. Three to nine independent experiments were performed quantifying from 3 to 8 cells each one. The graph shows the fluorescence quantification on MutuDC1940 surface incubated with H-ACs-P2X7 (black), MutuDCs pretreated with oATP (300μM), and incubated with H-ACs-P2X7 (red) and MutuDCs incubated with parental H-ACs (gray).

(D) Slopes calculated from the fluorescence quantification curves shown in (C) Data are represented as the mean ± SEM. Results were analyzed using non-parametric Mann-Whitney test.

(E) Ninety minutes after the challenge with H-ACs-P2X7 or H-ACs, cells were analyzed by confocal microscopy. Representative micrographs show MutuDCs expressing eGFP, CM labeled H-ACs and the superposition of both colors. A peripheral (white arrows) and vesiculated (red arrows) distribution pattern are indicated. Scale bar 10 µm. ∗∗ and ∗ represent p < 0.01 and p < 0.05 respectively.