Methods for the Measurement of Early Events in Toxoplasma gondii Immunity in Mouse Cells
Critical steps in resistance of mice against Toxoplasma gondii occur in the first 2 or 3 h after the pathogen has entered a cell that has been exposed to interferon γ (IFNγ). The newly formed parasitophorous vacuole is attacked by the IFNγ-inducible IRG proteins and disrupted, resulting in death of the parasite and necrotic death of the cell. Here we describe some techniques that we have used to describe and quantify these events in different combinations of the host and the parasite.
Key wordsToxoplasma gondii Parasitophorous vacuole Mouse Innate immunity IRG proteins Interferon-γ Diaphragm-derived cells Reactive necrosis Flow cytometry Immunofluorescence
Dulbecco’s modified Eagle’s medium
Fluorescence-activated cell sorting
Fetal bovine serum
Human foreskin fibroblasts = Hs27 cells
Heat-inactivated fetal bovine serum
Multichannel microscope slide
Multiplicity of infection
Nonessential amino acids
- P/W buffer
Permeabilization/wash buffer (for intracellular staining for FACS)
Relative centrifugal force
Toxoplasma major surface antigen 1 or P30
- T. gondii, Tg
The authors record their thanks to previous members of the laboratory who contributed to the development of the study of early postinfection events in T. gondii immunity and pioneered the application of several of these techniques. The present work would also not have been possible without the contributions of the service facilities of the IGC, in particular the Animal Facility, supported by the research infrastructure Congento, project LISBOA-01-0145-FEDER-022170, the Transgenics Facility, and the Antibody facility, both supported by Fundação Calouste Gulenkian, the Advanced Imaging Unit, supported by the project PPBI-POCI-01-0145-FEDER-022122 and the Flow Cytometry Unit, supported by the project LISBOA-01-0145-FEDER-007654.
This work was supported by central funds of the Instituto Gulbenkian de Ciência, by the Sonderforschungsbereiche 670 and 680 and Schwerpunkt 1399 of the Deutsche Forschungsgemeinde. Joana Loureiro received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie Grant Agreement number 708694 entitled “Toxoplasma Sensing.”
Author contributions: Subheading 1, Jonathan Howard; Subheading 2, Catalina Alvarez and Ben Mueller; Subheadings 3 and 4, Ana Lina Rodrigues and Joana Loureiro; Subheading 5, Joana Loureiro; Subheading 6, Claudia Campos. All authors contributed to the preparation and editing of the entire manuscript.
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