Fig. 4.

DiI-Ac-LDL uptake: Cell monolayers were exposed to fluorescent DiI-Ac-LDL and microscopically analyzed (A) in endothelial cells [HUVEC, HUVEC-hTERT, and fibroblasts (HSF)]. Cells were counterstained with calcein. Boxed areas are enlarged in the same row, rightmost column. Both HUVEC cell types displayed a homogenous pattern with all cells labeled with DiI-Ac-LDL (A a, A d). Detailed visualization revealed punctuate pattern in perinuclear regions (A c, A f). HSF showed no uptake of DiI-Ac-LDL (A g, A i). FACS analysis (B) resulted in more than 96.6% positive primary and immortalized HUVECs after incubating with DiI-Ac-LDL, whereas HSF showed no DiI-Ac-LDL uptake. Tube formation (C). Cells were cultured on Matrigel™ and double-stained with DiI-Ac-LDL and calcein [fluorescence images (C a, C b, C d, C e, C g, C h); corresponding phase contrast images (C c, C f, C i)]. Both HUVEC cell types resulted in capillary tube formation. These tubes were formed by intact endothelial cells, as shown by co-labeling with DiI-Ac-LDL (C a, C d) and calcein (C b, C e). In contrast, HSF were not able to form capillary-like tubes on Matrigel (C i) even though they were still viable (C h). *P < 0.05 with respect to untreated cells, scale bars 400 μm, merged details 100 μm