Shape Changes

Several aspects of phagocyte physiology, including motility, phagocytosis, translocation of granules, degranulation and recycling of receptors, and their regulation, are associated with changes in the organization of the cytoskeleton. Upon activation, changes in the cytoskeleton arrangements occur, and actin polymerization (G-actin assembly to form F-actin filaments) represents one of the most dynamic phenomena that characterize phagocyte activation (37). Actin polymerization may be measured by flow cytometry using phalloidin, a fluorescent peptide with a high affinity for polymerized actin (38,39). Both NBD (nitro blue diazonium)-phallacidin and fluorescein isothiocyanate (FITC)-phallacidin facilitate the measurement of the global amount of F-actin with high sensitivity. Flow cytometry, however, does not make a distinction between the two pools of F-actin molecules described as triton-soluble and triton-insoluble (40). Before staining with phalloidin, phagocytes have to be permeabilized.

Forward scatter (FSC) and SSC may also be used to detect shape changes. FSC is proportional to size, and when neutrophils are activated in suspension, they swell and exhibit increased FSC (41). Keller et al. (42) showed that there is a direct relationship between light scattering in flow cytometry and changes in shape, volume, and actin polymerization. Shape change has been used to measure the effect of a variety of chemokines on the polarization of eosinophils (43).

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