Ix83 fluoview1200

Live monocytes were stained with Alexa647-CTB and anti-Dectin-1 for 30 minutes at 4°C, then with Cy3-conjugated goat-anti mouse (Jackson ImmunoResearch), washed, pre-treated or not with MBCD (5 mM) or DAmb (5 μg/ml) and then incubated for 30 minutes on ice with FITC-conjugated HK C. albicans at 1:3 ratio, to allow cell-cell interaction without phagocytosis. Cells were then resuspended in warm DMEM without PhenolRed supplemented with 1% FBS and 400.000 cells/well were plated on 8-wells ibiTreat μ-slide (ibidi) for time lapse imaging and acquisition started 10–20 min after plating. Cells were recorded for 2 hours. Fluorescent and DIC images were acquired at the Olympus iX83 FluoView1200 equipped with Olympus CellVivo incubation system to control temperature, humidity and CO₂ conditions. The following settings were used: 60x, NA1,35 objective, acquisition mode sequential, no line average, zoom 3, single slice. Time series were acquired for 2 hours at 1 minute interval. Laser lines: 473 (0.4%), 559 (9%), 635 (15%).

For three-dimensional confocal microscopy analysis, 50-µm-thick sections were immunostained with anti-FABP4 antibody and images were acquired at 1024x1024px by an Olympus iX83 FluoView1200 laser scanning confocal microscope using an UPLSAPO60x oil NA1,35 (Olympus), 473 nm, 559 nm and 635 nm lasers and a z-step of 200 nm. The image stacks were visualized as three-dimensional (3D) reconstruction performed with Imaris v8.1.2 software (Bitplane). For 3D volume rendering, the “Surfaces” tool was used to generate 3D structures corresponding to P. falciparum gametocytes (red) and vessel (green); to highlight the 3D structures, DIC transmitted light and nuclei staining (purple) was shown using “Ortho slicer” feature.