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Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1β in Human Monocyte-derived Dendritic Cells
Published on: May 22, 2014
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Noncanonical NLRP3 Inflammasome Activation: Standard Protocols
Raíssa Leite-Aguiar1, Luiz Eduardo B Savio1, Robson Coutinho-Silva2
1Laboratory of Immunophysiology, Biophysics Institute Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
Methods in Molecular Biology (Clifton, N.J.)
|August 14, 2023
Summary
Analyzing inflammasome activation involves measuring cytokines like IL-1β and IL-18. Cleaved gasdermin D (GSDMD) offers an ideal pathway for studying noncanonical inflammasome activation.
Area of Science:
- Immunology
- Cellular Biology
Background:
- Inflammasomes are critical immune complexes involved in inflammation.
- Canonical inflammasome activation typically involves caspase-1, leading to IL-1β and IL-18 maturation.
- Assessing inflammasome activation traditionally relies on detecting mature cytokines or danger signals.
Purpose of the Study:
- To outline methods for assessing canonical and noncanonical inflammasome activation.
- To highlight the utility of gasdermin D (GSDMD) cleavage as a marker for noncanonical inflammasome activity.
Main Methods:
- Enzyme-linked immunosorbent assay (ELISA) and Western blots for cytokine and protein detection.
- Real-time PCR for measuring mRNA expression.
- Immunocytochemistry for protein localization.
- Analysis of cleaved gasdermin D (GSDMD) for noncanonical inflammasome assessment.
Main Results:
- Canonical inflammasome activation is indicated by extracellular mature IL-1β and IL-18.
- Noncanonical inflammasome activation can be assessed via similar techniques.
- Cleavage of gasdermin D (GSDMD) is a key event in noncanonical inflammasome activation and pyroptosis.
Conclusions:
- Multiple assays, including ELISA and Western blots, can differentiate active and inactive cytokine forms.
- Gasdermin D (GSDMD) cleavage provides a specific readout for noncanonical inflammasome activation.
- Studying GSDMD cleavage is a valuable approach for understanding noncanonical inflammasome pathways.
