PMID: 6406600May 27, 1983Paper

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

Journal of Immunological Methods
S SahlinI Rundquist

Abstract

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 of...Continue Reading

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Citations

Jun 4, 1993·Journal of Immunological Methods·C P WanB H Lau
Sep 14, 1994·Journal of Immunological Methods·N Plasman, B Vray
Oct 1, 1996·Veterinary Immunology and Immunopathology·K M LamJ P Eiserich
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Oct 2, 2012·Biomacromolecules·Danielle S W Benoit, Molly E Boutin
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Oct 1, 1993·Acta Physiologica Scandinavica·T Sundqvist, S M Liu
Jun 17, 2008·Applied and Environmental Microbiology·Bojana StojkovicSteven R Blanke
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Apr 13, 2005·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Jari Nuutila, Esa-Matti Lilius

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