Macrophage phagocytosis: use of fluorescence microscopy to distinguish between extracellular and intracellular bacteria.
One of the challenges of phagocytosis research is to differentiate bacteria adherent to a host cell from bacteria which the cell has internalized. To address this question, various techniques such as fluorescence microscopy, electron microscopy, and flow cytometry have been used. We have adapted a flow cytometric method (Fattorossi et al., 1989) to use fluorescence microscopy for studying phagocytosis of fluorescein-labeled Listeria by inflammatory mouse peritoneal macrophages. In this assay, ethidium bromide is used as a quenching agent and is added to cells after they have phagocytosed labeled bacteria. Ethidium bromide causes extracellular FITC-labeled Listeria to fluoresce red-orange, whereas intracellular bacteria are not exposed to the dye and remain green. This process allows distinction between intracellular and extracellular bacteria by simultaneous visualization of both populations.
Department of Medicine, National Jewish Center for Immunology and Respiratory Medicine, Denver, CO 80206.
D A
DA
Drevets
P A
PA
Campbell
D000818:Animals; D001422:Bacterial Adhesion / Q000276:immunology*; D004705:Endocytosis / Q000276:immunology; D005260:Female; D008089:Listeria monocytogenes / Q000166:cytology / Q000276:immunology; D008264:Macrophages / Q000166:cytology / Q000276:immunology*; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D008856:Microscopy, Fluorescence; D010587:Phagocytosis / Q000276:immunology*