DETERMINATION OF QUANTITATIVE PARAMETERS OF ESCHERICHIA COLI PHAGOCYTOSIS MOUSE PERITONEAL MACROPHAGES
V. V. Miliukiene,1,* G. J. Biziulevieiene,2,3 L. P. Chaustova,1
A. V. Pilinkiene,3 G. A. Biziulevieius 3
1 Institute of Biochemistry, Vilnius, 2 Faculty of Natural Sciences, University of Vilnius,
and 3 Institute of Immunology, University of Vilnius, Lithuania;
* e-mail: valem@bchi.it
The fluorometric method was used to study guantitative parameters of phagocytosis of fluorescein-labeled
Escherichia coli cells by mouse peritoneal macrophages. Å. coli cells were conjugated with
fluoresceinisothiocyanate (FITC) and then incubated with macrophages. At the end of the assay phagocytosis was
arrested with a lysing solution (0.5 % Triton X-100 in 0.01 M phosphate-buffered 0.15 M saline, pH 7.4). Trypan blue
at a concentration of 0.04 % was used as a quenching agent to differentiate between attachment and ingestion of
Å. coli cells. The time course analysis within this method showed that phagocytosis of E. coli cells was
temperature and opsonin dependent. The number of Å. coli cells ingested by macrophages increased rapidly during
the initial 60 min of incubation at 37 °Ñ. Å. coli cells required opsonization with 5 % native serum to achieve
their optimal uptake. The uptake of nonopsonized bacteria by macrophages was significantly lower that that of
opsonized ones (P < 0.05). It was demonstrated that sodium azide inhibited phagocytosis of Å. coli cells by
mouse peritoneal macrophages in a dose-dependent manner.
Key words: Escherichia coli, macrophages, phagocytosis, fluorometric assay, quantitative
parameters
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