Phagocyte C3-mediated attachment and internalization: flow cytometric studies using a fluorescence quenching technique

Abstract

The dynamics of phagocyte C3-mediated attachment and internalization of fluorescein-isothiocyanate (FITC)-labelled zymosan particles was studied by a flow cytometric (FCM) fluorescence quenching technique, using trypan blue as quenching agent. Trypan blue effectively quenched the fluorescence of extracellular, i.e. free and phagocyte-attached, zymosan particles, but did not influence on the fluorescence of particles internalized by phagocytes. During phagocytosis, an average of 2 C3-coated zymosan particles were simultaneously attached to the phagocyte surface, and the number of attached particles could not be increased by increasing the zymosan to leukocyte ratio, the concentration of C3, the incubation time, or by inhibiting internalization by Cytochalasin B. Phagocyte C3-mediated internalization of zymosan particles was dependent on the concentration of complement, and in the presence of sufficient amounts of C3, internalization continued until saturation was reached at 11 particles per phagocyte.