Sp8 690 fluorescence microscope

HUVECs were grown as non-stimulated or TNF-α-stimulated monolayer for 18 h on circular coverslips coated with fibronectin as described above. Chemically synthesized human CCL2 and CCL5 were supplied with site-specific biotinylation (Almac). 120 nM chemokine was added to the cells for 30 min at 37°C in 5% CO₂, after washing with Vascular Cell Basal Medium, cells were incubated with Alexa Fluor 594 streptavidin (1:500, 405240; BioLegend) for an additional 30 min. Cells were washed and fixed with 4% paraformaldehyde and after counter stained with DAPI images were captured using Leica SP8 (690) fluorescence microscope and analyzed using IMARIS software.

Circular cover slips (12 mm diameter) were placed in wells of a 24-well tissue culture plate and coated with recombinant human fibronectin for 2 h at 37°C or overnight at 4°C. Cells were grown on these cover slips for 18–20 h in complete vascular cell growth medium at 37°C with 5% CO₂. Samples were fixed with paraformaldehyde (4%, Thermo Scientific)) in PBS and 2% sucrose for 30 min at RT. For permeabilization, samples were incubated with 0.1% Triton X-100 for 5 min and after extensive washing, blocked with 2% BSA/PBS for 30 min at RT. Samples were incubated with primary antibodies against chemokines CCL2 (5 μg, MAB679) CCL5 (5 μg, MAB278), CCL7 (5 μg, MAB282), CCL8 (5 μg, MAB281), CCL20 (5μg, AF360), CXCL9 (5 μg, MAB392), mouse IgG₁ isotype control (5 μg, MAB002) and mouse IgG_2B isotype control (MAB004) all from R&D Systems, at RT for 2 h. Cells were washed three times with PBS before incubating with secondary antibody Alexa Fluor 488 (1:500, A-11017; Invitrogen) for 1 h and after washing three times with PBS, were counter-stained with DAPI (1μg/ml 62248; Invitrogen). Images were captured using a Leica SP8 (690) fluorescence microscope. Images were obtained with 40x objective. Images were analyzed using IMARIS software (Oxford Instruments).