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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

Measurement of active phagocytosis by polymorphonuclear leukocytes by fluorescence liberation from phagocytized microspheres.

Phagocytosis by polymorphonuclear leukocytes (PMN) was determined by a newly developed technique based on measurement of liberation of a fluorescence substance from PMN phagosomes; 4-methylumbelliferyl-beta-D-glucuronide (4MUGL), which is a substrate of beta-glucuronidase in lysosome, was conjugated with a microsphere, and 4-methylumbelliferone (4MU) liberated from phagocytized 4MUGL-microspheres was measured. The microspheres were composed of glyceryl-methacrylate having a diameter of 2.0 micron. Liberating activity of six kinds of 4MUGL-microspheres containing various amounts of amino and carboxyl groups was compared. Among these six kinds of 4MUGL-microspheres, four kinds showed activity similar to that of morphological phagocytosis. These four kinds of 4MUGL-microspheres liberated 4MU into the extracellular fluid from PMN during phagocytosis. Furthermore, they were recognized as a substrate of purified beta-glucuronidase. 4MUGL-MS610 showed the highest liberating activity among the four kinds of microspheres. Optimal conditions for phagocytosis by PMN were determined using 4MUGL-MS610. Total liberation of 4MU from the microspheres increased almost linearly with incubation time with PMN from 0 to 60 min and was linear with 4MUGL-MS in concentrations up to 4 X 10(8) microspheres/ml. This liberation was parallel to phagocytosis in a dose-dependent fashion. During 10-min incubation 20.4% of 4MU was liberated from 4MUGL-microspheres with phagocytosis. Seventy-five percent of the liberated 4MU was distributed in the extracellular fluid. 4MU distributed in the extracellular fluid was not attributable to hydrolysis of unphagocytized microspheres by beta-glucuronidase extracellularly leaked from PMN by phagocytosis. Also phagocytized 4MUGL-MS610 by PMN was observed by scanning electron microscopy. These results indicate that 4MU was liberated from 4MUGL-MS by hydrolysis due to beta-glucuronidase released into phagosomes with phagocytosis by PMN. Sensitivity of this assay was limited to about 50 pmol/ml, being less than 0.5-1 microsphere phagocytized into one cell.[1]


  1. Measurement of active phagocytosis by polymorphonuclear leukocytes by fluorescence liberation from phagocytized microspheres. Suzuki, K., Uchida, T., Sakatani, T., Sasagawa, S., Hosaka, S., Fujikura, T. J. Leukoc. Biol. (1986) [Pubmed]
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