Fig. 2.

Sprouting assay validation. (A) Confocal imaging of LEC bead coverage by immunofluorescent CD31 staining (red), cytosolic labeling with CTG dye (green), and nuclear labeling with Hoechst dye (blue) demonstrates a dense LEC population with characteristic cell-cell contacts in a maximum intensity projection (left panel) and optical cross-sections (central panel). (B) Fluorescence images of CTG-labeled sprouting LECs show the existence and visualization of sprouts in all scanned layers. (C) Sprouting response as sprout number per bead measured for different combinations of lymphangiogenic stimulants in the sprouting assay. A combination of 40 ng/ml VEGF-A, 40 ng/ml bFGF, and 2 μM S1P yielded the strongest sprouting response that was inhibited after addition of the receptor tyrosine kinase inhibitor sunitinib (5 μM). (D) Representative images of sprouting beads from panel C show pronounced CTG positive protrusions in S1P+VEGF-A+bFGF treated wells but not in S1P+VEGF-A+bFGF+sunitinib or untreated wells. */**/*** p < 0.05/0.01/0.001 in one-way ANOVA with simple “contrast” post test. +/++/+++ p < 0.05/0.01/0.001 in unpaired Student t-tests. n = 10. Bars show mean ± SEM. All scale bars represent 30 μm.