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Measuring Innate Immune Responses to Bacterial Viability

  • Julien Moretti
  • Nicolas Vabret
  • J. Magarian BlanderEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1714)

Abstract

The innate immune system directly senses microbial viability via the detection of a special class of viability-associated pathogen-associated molecular patterns (vita-PAMPs), such as prokaryotic messenger RNA. In the case of Gram-negative bacteria, detection of bacterial viability by phagocytes leads to a unique activation of inflammasome and type I interferon pathways, resulting in a robust pro-inflammatory innate response and a vigorous adaptive immune response. This protocol describes the methods required to study activation of both inflammasome and type I interferon pathways after stimulation of mouse bone marrow-derived macrophages with live or killed Gram-negative and Gram-positive bacteria. It covers the generation and handling of bone marrow-derived macrophages, the culture and killing of bacteria, the preparation of bacterial messenger RNA, and the stimulation of macrophages with live or killed bacteria. Lastly, this protocol describes the techniques employed to measure the hallmarks of inflammasome (secretion of interleukin-1β) and type I interferon (activation of TBK1, IRF3 and secretion of type I interferon) pathways.

Keywords

Vita-PAMP Bone marrow-derived macrophages Inflammasome Interleukin-1-β Type I Interferon TANK-binding kinase 1 

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Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Julien Moretti
    • 1
    • 2
  • Nicolas Vabret
    • 3
  • J. Magarian Blander
    • 1
    • 2
    • 4
    Email author
  1. 1.Jill Roberts Institute for Research in Inflammatory Bowel DiseaseWeill Cornell MedicineNew YorkUSA
  2. 2.Joan and Sanford I. Weill Department of MedicineWeill Cornell MedicineNew YorkUSA
  3. 3.Department of MedicineIcahn School of Medicine at Mount SinaiNew YorkUSA
  4. 4.Department of Microbiology and ImmunologyWeill Cornell MedicineNew YorkUSA

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