Abstract
Toxoplasma gondii is an obligate intracellular protozoan parasite that causes the disease toxoplasmosis. Chronic infection is established through the formation of tissue cysts predominantly in cardiac and neurologic tissues. A defining characteristic of T. gondii is its ability to evade the host’s immune defenses; specifically, T. gondii can invade and persist within host phagocytes, using them to disseminate to the brain and central nervous system where cysts are then formed. This protocol is used to evaluate the ability of Toxoplasma gondii to survive and replicate within naive and activated murine bone marrow-derived macrophages at the level of single infected cells. In the following protocol macrophages are naive or activated with IFN-γ and LPS but different activation stimuli can be utilized as well as different host cell populations and diverse inhibitors. Parasite replication is determined by evaluating the number of parasites per vacuole over time using immunofluorescence staining for parasties and microscopic analysis. Kinetic determination of parasite number per vacuole accurately reflects parasite replication over time as vacuoles-containing parasites do not fuse with one another. Isolation of murine bone marrow-derived macrophages, preparation of conditioned L929 cells for collection of macrophage colony-stimulating factor, and staining for fluorescence microscopy included in the protocol has broad applicability. This protocol works well for pathogens like Toxoplasma gondii that reside in vacuoles that do not fuse with one another and that can be visualized by microscopy.
Keywords: Toxoplasma, Macrophages, Cell-autonomous immunity, Nitric oxide
Materials and Reagents
Note: source of reagents is not critical to experiment.
Equipment
Procedure
Recipes
Protocol for L929 conditioned media
Protocol for bone marrow isolation and bone marrow macrophage differentiation
Acknowledgments
This work was adapted from a protocol we used for studies published in Skariah et al. (2012). The studies were funded by a grant to D.G. Mordue from the National Institute of Health (NIH) 1R01 AI 072028.
References
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