Dil ac ldl

Dil fluorescent dye-labeled acetylated low-density lipoprotein (Dil-ac-LDL; 10 μg/mL, Cell Applications) was added to phase IV medium of iECs at day 11 or day 21 and incubated for 4 h at 37°C, as described previously (24 (link)). Cells were then washed with PBS, fixed, and stained with DAPI. Images were taken with ImageXpress Micro XLS (Molecular Devices) and analyzed using ImageJ software. HUVECs fed with phase IV medium were used as positive control, and iPSCs fed with mTeSR⁺ medium were used as negative control.

Endothelial cells were incubated with Dil-Ac-LDL (1:100) (Cell Applications, San Diego, CA, USA) in complete media overnight, and plated in EBM-2 complete media for 5–6 h incubation in pre-coated Matrigel suspension wells (1:2 dilution with PBS). The endothelial cells used in co-culture with MDA-MB-468 and PC-3 cancer cells were transfected with LV-EF1α-tdTOMATO-IRES-Puro, pre-made lentivirus, which expresses tdTomato (SignaGen Laboratories, Frederick, MD, USA). We followed the manufacturer’s protocol to perform the transfection and induce tdTomato expression. Cancer cells were incubated with 1 μM of CellTrace green (CFSE, Life Technologies, Waltham, MA, USA) in DMEM basal media for 1 h. The cells were added to the preformed vessels in their respective complete media in a 1:1 ratio, which was the same as the ratio of cancer cells: endothelial cells and incubated for 24 h before analysis.

Freshly harvested bone marrow mononuclear cells were seeded onto polymer coverslip-bottom chamber slides (Ibidi) coated with fibronectin (Sigma-Aldrich). After two days, nonadherent cells were removed with a PBS wash. Media was changed and cells were cultured for a total of seven days. On day six of culture, media was changed to endothelial growth media with 1.5% FBS to serum-starve the cells. On day seven of culture, cells were incubated with 10 mg/L of Dil-Ac-LDL (Cell Applications) for 4 h at 37 °C. Cells were washed and fixed with 4% paraformaldehyde, and counter-stained with DAPI. Cells were imaged with confocal microscopy and images were analyzed with FIJI to determine the number of cells with incorporation of DiI-labeled Ac-LDL.