Characterization of ICAM-1GFP iHUVECs. (A) Surface expression of ICAM-1 on 4-hour TNF-α-activated ICAM-1GFP iHUVECs (red lines) was assessed by indirect immunofluorescence staining and flow cytometry and compared with that of 4-hour (left) and 24-hour TNF-α-activated iHUVECs (right; black solid line). Dotted line indicates nonbinding mAb. (B) iHUVECs or ICAM-1GFP iHUVECs were treated with TNF-α for 4 hours or 24 hours or media alone (0 hours), as indicated. Expression of endogenous ICAM-1 and ICAM-1GFP was determined by immunoprecipitation of biotinylated ICAM-1 on cell surface, as described in “Materials and methods.” (C) Surface distribution pattern of ICAM-1 on 4-hour TNF-α-activated iHUVECs and ICAM-1GFP iHUVECs was detected by live-cell epifluorescence microscopy (× 40 objective; top) and confocal microscopy (× 60 objective; bottom) as described in “Materials and methods.” Orthogonal views of endothelium were reproduced from serial z-sections horizontally scanned using confocal microscopy (0.5-μm increments) and manipulated with Lasersharp 2000 V4.3. To visualize ICAM-1, live iHUVECs were stimulated with TNF-α for 4 hours and incubated with Alexa 488-labeled anti-ICAM-1 mAb CL23.4, washed, and observed by confocal microscopy. ICAM-1GFP on 4-hour TNF-α-activated ICAM-1GFP iHUVECs was detected by GFP signal (not Alexa 488 anti-ICAM-1 mAb). (D) Live confluent HUVECs, iHUVECs, and ICAM-1GFP iHUVECs were stained with Alexa 568-labeled anti-JAM-A mAb 1H2A9 or anti-VE-cadherin TEA1/31. Images were obtained by the live-cell epifluorescence imaging as detailed in “Materials and methods.”