Real-time imaging of the LFA-1 activation in whole human blood flow chambers. (A-B) Whole blood was treated with Alexa Fluor 594–conjugated KIM127 (5 μg/mL). After 5 minutes, whole blood was perfused through flow chambers coated with E-selectin. Fluorescence intensity was detected by the epifluorescence microscope with the enhancer. (B) Fluorescence intensity reflecting KIM127 binding was measured at 2-second intervals for representative cells (■, □, ▼, ♦, ○, ▲, +, ×) and normalized to the maximal intensity (= 1). Data were averaged at 4-second intervals (indicated by ● joined by line; mean ± SEM). Data representative of 2 independent experiments. (C) Whole blood was treated with Alexa Fluor 594–conjugated antibody (5 μg/mL) and perfused through flow chambers coated with E-selectin. After saturation, IL-8 was added to whole blood, and the change of the Alexa Fluor 594 intensity was measured. Data are presented as mean ± SEM and are representative of 3 independent experiments.