Quantification of F-actin staining in 24-hour TNF-α–activated ECs on neutrophil adherence.

F-actin was detected by rhodamine-phalloidin stain as described, and the image was scanned and monitored using a Leica TCS-NT laser scanning confocal microscope. The mean pixel intensity of the F-actin fluorescence per field (A) and the percentage of the field (1000×) occupied by F-actin staining (B) were measured. Data are expressed as percentage changes over the corresponding control (exposing to buffer for the same duration) and presented as mean ± SEM from at least 10 images for each slide. The experiment was repeated 2 times with similar results. *Mean pixel intensity and percentage area in ECs in response to adherent neutrophils were significantly higher compared to addition of buffer for the same duration (P < .05).