title: Determination ofToxoplasma gondiiReplication in Naïve and Activated Macrophages.
abstract: Toxoplasma gondiiis 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 ofT. gondiiis its ability to evade the host's immune defenses; specifically,T. gondiican 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 ofToxoplasma gondiito 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 likeToxoplasma gondiithat reside in vacuoles that do not fuse with one another and that can be visualized by microscopy.
authors.affiliation: Department of Microbiology and Immunology, New York Medical College, Valhalla, USA.
authors.forename: Emma
authors.initials: E
authors.lastname: Iaconetti
authors.affiliation: Department of Microbiology and Immunology, New York Medical College, Valhalla, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Lynch
authors.affiliation: Department of Microbiology and Immunology, New York Medical College, Valhalla, USA.
authors.forename: Nathaniel
authors.initials: N
authors.lastname: Kim
authors.affiliation: Department of Microbiology and Immunology, New York Medical College, Valhalla, USA.
authors.forename: Dana G
authors.initials: DG
authors.lastname: Mordue