Differentiation between attached and ingested immune complexes by a fluorescence quenching cytofluorometric assay.

Immune complexes attached to and ingested by human polymorphonuclear (PMN) cells were quantified by cytofluorometry using a fluorescence quenching assay which permits differentiation between attachment and ingestion. The fluorescence intensity decreased after ingestion as a result of the low pH in the phagolysosomes. When extracellular pH was lowered a slight decrease in phagolysosomal pH was detected in macrophages but not in PMN. When measuring total fluorescence, interaction at pH 5.8 for PMN and at pH 4.4 for macrophages is recommended, since the intensity of extra- and intracellular fluorescence are equal under these conditions. Thirty different dyes were tested for dye exclusion and fluorescence quenching of FITC-conjugated yeast particles, and FITC-conjugated IgG. Because of the lysosomotropic effect of basic dyes, acid and direct dyes are preferable as quenching agents. We could not find physical or chemical properties of the dyes that correlated with their quenching effect. Heat aggregated IgG was used as an immune complex analogue in the development of the assay. Trypan blue (0.2 mg/ml) at pH 4.4 was found to be the best quenching agent of extracellular fluorescence when using ingested aggregated IgG. The technique offers a simple method of quantifying ingested protein aggregates and of studying heterogeneity in phagocyte populations.