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Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages
Published on: May 21, 2018
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A comprehensive guide to studying inflammasome activation and cell death
Rebecca E Tweedell1, R K Subbarao Malireddi1, Thirumala-Devi Kanneganti2
1Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA.
Nature Protocols
|September 8, 2020
Summary
This study presents a comprehensive protocol for assessing inflammasome activation and cell death. The method uses in vitro and in vivo strategies to reliably measure key indicators of the innate immune response.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Inflammasomes are critical protein complexes in innate immunity, responding to infection and injury.
- Inflammasome activation involves sensor oligomerization and downstream effector functions, leading to cell death.
- Assessing inflammasome activation requires reliable experimental readouts.
Purpose of the Study:
- To describe a comprehensive protocol for activating inflammasomes.
- To detail methods for assessing inflammasome activation and cell death pathways.
- To provide a consistent workflow for evaluating innate immune responses.
Main Methods:
- Utilized in vitro (bone marrow-derived macrophages) and in vivo (mice) models for inflammasome activation.
- Assessed inflammasome activation through ASC speck formation, caspase-1 and gasdermin D cleavage.
- Measured IL-1β, IL-18, caspase-1, and LDH release, alongside real-time cell death imaging.
Main Results:
- Developed a protocol enabling parallel assessment of multiple downstream inflammasome effects.
- The workflow allows for unequivocal determination of inflammasome activation and cell death status.
- Analyses are completed within approximately 24 hours.
Conclusions:
- The presented multifaceted approach offers a comprehensive and consistent protocol.
- This method reliably assesses inflammasome activation and associated cell death pathways.
- The protocol is valuable for studying innate immune responses in infection and sterile inflammation.
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