Optical and fluorescence detection of neutrophil integrin activation

Neutrophils are among the first cells to respond to acute inflammation through a multistep process initiated by selectin mediated rolling, which transitions to an integrin/intercellular adhesion molecule-dependent arrest and transmigration across endothelium. A conformational shift in the CD11/CD18 adhesion receptor on neutrophils is a critical determinant of the efficiency of recruitment on inflamed endothelium. For instance, beta2-integrin expression level is upregulated up to 10-fold by fusion of cytoplasmic granule pools of CD11b/CD18 (Mac-1). Furthermore, a rapid increase in affinity and membrane clustering of CD11a/CD18 (LFA-1) is necessary for efficient deceleration and arrest in shear flow. We present methods here to quantify the changes in receptor expression and affinity that support neutrophil adhesive phenotypes. Techniques involving real-time fluorescence flow cytometry and parallel plate rheometry coupled with light microscopy are presented.