Calcein am

HMVECs were grown to 80% confluency (1 × 10⁶ cells/ml) in 10-cm dishes. Cells were then transduced as described previously (m.o.i. = 20). The following day, transduced cells were serum-deprived by replacing complete growth medium with EBM-2MV for 12 h. Next, cells were resuspended in either EBM-2MV medium supplemented with 0.4% FBS or EBM-2MV medium supplemented with 0.4% FBS containing 20 ng/ml VEGF. For VEGF signaling inhibition, cells were preincubated with SU1498 (25 μm) for 2 h in EMB-2MV before seeding. Cells were seeded at a density of 2 × 10⁴ cells/, BD Biosciences). The plates were incubated for 18 h in a 37 °C incubator with 5% CO₂. After incubation, cells were stained with CalceinAM, and images were captured using fluorescent microscopy (Olympus). Tube-like network structures were analyzed using AngioTool analysis software (25 (link), 26 (link)). All treatments were performed in triplicate and repeated at least five times.

PI (Calbiochem) and calcein-AM (Invitrogen) uptake were used to evaluate hBMEC and Vero E6 cell viability. ZIKV-infected (MOI, 10) or mock-infected hBMECs were seeded into 96-well plates and at the indicated times costained by the membrane-permeable dye calcein-AM (3 μM; green fluorescence in live cells), and 2.5 μM propidium iodide (red fluorescent DNA stain to detect dead cells). Images of calcein-AM-positive versus PI-positive cells were resolved using an Olympus IX51 microscope and Olympus DP71 camera and overlayed using Adobe Photoshop.

Human CCD-18Co cells were incubated with berberine, 1 h before addition of OSM (10 ng/ml) for 24 h. Human U937, THP-1, and Jurkat T cells were stained with 1 μM calcein AM (BD Biosciences) for 30 min at room temperature and then added onto CCD-18Co monolayer cells and adhered for 30 min at 37 °C in a humidified incubator of 5% CO₂. Non-adherent cells were washed away with PBS and the calcein AM-labeled cells with green fluorescence were monitored using a fluorescent microscope (Olympus IX73).
For in vitro chemotaxis, the above supernatants from OSM-stimulated CCD-18Co cells were plated into the lower chamber of trans-well plates (Corning Incorporated, Corning, NY, USA), meanwhile, calcein AM-labeled U937, THP-1, and Jurkat T cells were added into the upper chamber for 2 h incubation. Subsequently, cells in the lower chamber were amounted using hemocytometer (Thermo Fisher Scientific) and photographed under the fluorescent microscope (Olympus IX73).

Human umbilical vein endothelial cells (HUVECs) in EBM-2 media supplemented with 0.1% FBS were plated on Matrigel basement membranes (BD Biosciences, NJ, USA) and incubated for 8 h under hypoxic conditions. Auraptene was added to media, after which cells were stained with calcein-AM (Sigma-Aldrich, SG, Switzerland). Intracellular calcein-AM fluorescence (n = 10 slides/condition) was imaged using an IX70 fluorescence microscope (Olympus, Tokyo, Japan).