Time-dependent changes of CXCR-4 expression and localization during ECM-dependent endothelial cell tube formation.
HUVECs (10 × 103) were plated on Matrigel-coated, glass chamber slides (2-well) and were incubated at 37°C for 1, 2, 4, and 16 hours. After incubation, cells were fixed with 1% formaldehyde and examined unstained with a phase-contrast microscope or stained for CXCR-4 with a murine monoclonal antihuman CXCR4 antibody (IgG2B, clone 44716.111) followed by Alexa 568–conjugated goat antimouse IgG, and they were examined with an epifluorescence microscope with or without a confocal system. Representative images from phase-contrast microscopy showed different stages of HUVEC migration and assembly into tubular structures at 1, 2, 4, and 16 hours. Parallel changes in levels and distribution of CXCR-4 surface expression on HUVEC detected by epifluorescence (original magnification, ×20 all time points except ×10 at 16-hour time point) and confocal microscopy (original magnification, ×60).
