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Methods for the Measurement of Early Events in Toxoplasma gondii Immunity in Mouse Cells

  • Catalina Alvarez
  • Ana Claudia Campos
  • Jonathan C. HowardEmail author
  • Joana Loureiro
  • Urs Benedikt Müller
  • Ana Lina Rodrigues
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2071)

Abstract

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 words

Toxoplasma gondii Parasitophorous vacuole Mouse Innate immunity IRG proteins Interferon-γ Diaphragm-derived cells Reactive necrosis Flow cytometry Immunofluorescence 

Abbreviations

CO2

Carbon dioxide

DMEM

Dulbecco’s modified Eagle’s medium

EDTA

Ethylenediaminetetraacetic acid

FACS

Fluorescence-activated cell sorting

FBS

Fetal bovine serum

H2O2

Hydrogen peroxide

HEPES

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid)

HFFs

Human foreskin fibroblasts = Hs27 cells

HI-FBS

Heat-inactivated fetal bovine serum

IFNγ

Interferon (IFN)-gamma

MMS

Multichannel microscope slide

MOI

Multiplicity of infection

NEAA

Nonessential amino acids

P/W buffer

Permeabilization/wash buffer (for intracellular staining for FACS)

PBS

Phosphate-buffered saline

PFA

Paraformaldehyde

RCF

Relative centrifugal force

RT

Room temperature

SAG1

Toxoplasma major surface antigen 1 or P30

Sta

Staurosporine

T. gondii, Tg

Toxoplasma gondii

WD

Working dilution

Notes

Acknowledgments

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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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Catalina Alvarez
    • 1
  • Ana Claudia Campos
    • 1
  • Jonathan C. Howard
    • 1
    • 2
    Email author
  • Joana Loureiro
    • 1
  • Urs Benedikt Müller
    • 2
  • Ana Lina Rodrigues
    • 1
  1. 1.Instituto Gulbenkian de CiênciaOeirasPortugal
  2. 2.Institute for GeneticsCologneGermany

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