Cellvivo incubation system

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%).

To analyze cell motility, A-549 cells were seeded onto the Ibidi-silicone insert on a cover glass-bottom 24-well plate for live cell imaging and incubated for 24 h. Subsequently, the inserts were dislodged, the cellular debris was removed by washing with RPMI, and the cells were incubated with different concentrations of PCH-1, and/or 2.5 ng/ml TGF-β1 (Sigma-Aldrich) in an imaging chamber (cellVivo incubation system, Olympus) at 37 °C with 5% CO₂. Images were captured every 10 min for 20 or 42 h under × 10 magnification using a fluorescence microscope (IX83 Inverted Microscope, Olympus) connected to an XC50 digital color camera (Olympus). The percentage of wound closure was quantified with ImageJ software.

hTERT-HSCs were seeded at 10,000 cells per well of a 48-well plate, allowed to adhere overnight and then transfected as described above. Following transfection, a scratch was made in each well and fresh medium was added with or without treatments and imaged every h for 48 h in the cellVivo incubation system by Olympus at 37 °C, 5% CO₂ in a humidified chamber. Data were analyzed in ImageJ using the “Wound_healing_size_tool” (author: Volker Baecker), which allows batch analysis of the stack of 48 images for each condition.