A new simple fluorometric assay for phagocytosis.
A highly sensitive, but simple and quantitative, fluorometric assay method for phagocytosis by cells such as macrophages and polymorphonuclear leukocytes was developed by utilizing fluorescent particles. Escherichia coli, Serratia marcescens, yeast, and latex particles were conjugated with fluorescein isothiocyanate and used as fluorescent particles. The assay procedure requires phagocytic cells, appropriate medium, fluorescent particles, sodium dodecyl sulfate, microtiter culture plate (24 wells), clinical centrifuge, and fluorescence spectrophotometer. One hundred assays can be done within 30 min after the incubation period. A time course analysis with this method showed that the phagocytosis of all these particles was dependent on temperature, and that the number of particles ingested by cells increased rapidly during the initial 30 min of incubation at 37 degrees C. Free fluorescent particles can be removed effectively by aspiration from the well. At 0 degree C, very few particles were ingested by cells or adsorbed onto the phagocytic cell surface as confirmed by fluorescence microscopy. An inhibitory effect of cepharanthin and sodium azide on phagocytosis was also confirmed by this method. The differential susceptibility of E. coli B and S. marcescens to phagocytosis also could be determined by this method.
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D001386:Azides / Q000494:pharmacology; D044182:Benzylisoquinolines; D004926:Escherichia coli / Q000276:immunology; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D007158:Immunologic Techniques; D007700:Kinetics; D008264:Macrophages / Q000502:physiology*; D008297:Male; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D008863:Microspheres; D009504:Neutrophils / Q000502:physiology*; D009895:Opsonin Proteins; D010587:Phagocytosis / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000276:immunology; D012706:Serratia marcescens / Q000276:immunology; D019810:Sodium Azide; D013050:Spectrometry, Fluorescence / Q000379:methods; D052117:Benzodioxoles
D000470:Alkaloids; D001386:Azides; D044182:Benzylisoquinolines; D006861:Hydrogen Peroxide; D009895:Opsonin Proteins; D019810:Sodium Azide; C006947:cepharanthine; D052117:Benzodioxoles