Trypsinized HUVEC were incubated in suspension, under plate rotation for 30 min at 37°C and 5% CO2, with 3.5 μm diameter magnetic beads conjugated with a biotinylated mouse anti-human VEGF Receptor 2 (from MACSiBeads, Miltenyi, Auburn, CA), at a ratio of beads to cells of 20:1. The cells were then fixed, permeabilized as described, stained with AlexaFluor 488-Phalloidin and further incubated with an anti-mouse-AlexaFluor 543 antibody for detection of the beads, and with 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride, 4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI, Sigma-Aldrich, St. Louis, MO) for visualization of nuclei.
Scaffolds containing live cells were also incubated with PKH26 (Sigma-Aldrich, St. Louis, MO), a hydrophobic red fluorescent label [28 (link)], dissolved either in the solution provided by the manufacturer to label the SFMs, or in phosphate buffer saline (PBS, pH 7.4), to induce its precipitation and to be phagocytosed by the cells.
Scaffolds containing live cells were also incubated with PKH26 (Sigma-Aldrich, St. Louis, MO), a hydrophobic red fluorescent label [28 (link)], dissolved either in the solution provided by the manufacturer to label the SFMs, or in phosphate buffer saline (PBS, pH 7.4), to induce its precipitation and to be phagocytosed by the cells.