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.
Schaff
Ulrich Y
UY
Department of Biomedical Engineering, University of California, Davis, CA, USA.
Sarantos
Melissa R
MR
Ting
Harold
H
Simon
Scott I
SI
D000954:Antigens, Surface / Q000032:analysis; D000939:Epitopes; D005453:Fluorescence; D016023:Integrins / Q000378:metabolism*; D008954:Models, Biological; D018375:Neutrophil Activation / Q000502:physiology*; D009504:Neutrophils / Q000276:immunology / Q000378:metabolism*; D055095:Optics and Photonics
D000954:Antigens, Surface; D000939:Epitopes; D016023:Integrins