Flow microfluorometric analysis of phagocyte degranulation in bacteria infected whole human blood cell cultures

摘要

A quantitative flow microfluorometric method was used to study the intensity of human blood phagocyte degranulation in response to viable Staphylococcus aureus or Yersinia pestis (vaccine strain EV) cells. Microorganisms were added directly to defibrinated whole blood. Uninfected (control) and infected blood samples were incubated at 37°C to 8 h. The results were recorded in dynamics after the staining of whole blood (0,1 ml) with acridine orange solution. Lymphocytes with a low azurophilic granule per cell content were discriminated from phagocytes by the measurement of single cell red cytoplasmic granule fluorescence. 30 000 cells in each sample were examined. S. aureus cells caused a dose-dependent decrease in the number of phagocytes having a high red cytoplasmic fluorescence intensity and a corresponding increase in the weakly fluorescent cell population (lymphocytes + degranulated phagocytes). In the presence of an initial S.aureus-to-phagocyte ratio more than 1:1, degranulation was measured after 3 h of incubation and to 8 h the percentage of degranulated phagocytes was at least 100 %. Y.pestis cells grown for 48 h at 28°C caused at same conditions the degranulation only about 50 % of cells. Y.pestis EV cells preincubated in broth for 12 h at 37°C did not stimulate the phahocyte degranulation. The results of these stadies suggest that analysis of cell populations via flow microfluorimeter technology may be a powerful tool in analysis bacterial infection.