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. 2018:1857:125-134.
doi: 10.1007/978-1-4939-8754-2_12.

Analyzing Necroptosis Using an RIPK1 Kinase Inactive Mouse Model of TNF Shock

Matija Zelic  1 , Michelle A Kelliher  2
Affiliations

Analyzing Necroptosis Using an RIPK1 Kinase Inactive Mouse Model of TNF Shock

Matija Zelic et al. Methods Mol Biol. 2018.

Abstract

The serine/threonine kinase RIPK1 has numerous biological and pathological functions, mediating prosurvival as well as prodeath apoptotic and necroptotic signaling pathways downstream of various receptors, including death receptors and Toll-like receptors (TLRs). RIPK1 has been implicated in various diseases, including ischemia-reperfusion injury and inflammatory bowel disease (IBD). The recent generation of RIPK1 kinase inactive mice has enabled us to genetically interrogate the role of RIPK1 kinase-mediated necroptosis in disease models. Here, we describe procedures utilizing kinase inactive Ripk1D138N/D138N mice to analyze necroptosis induction in vitro in bone-marrow derived macrophages (BMDMs) and in vivo in a murine model of TNF-induced shock.

Keywords: Bone marrow-derived macrophages; Caspase inhibitor; Cell death; Lipopolysaccharide; Necroptosis; TNF shock.

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Figures

Fig. 1
Fig. 1
RIPK1 kinase inactive BMDMs are resistant to LPS/zVAD-induced necroptosis. Primary BMDMs isolated from WT, Ripk1D138N/D138N and Ripk3−/− mice were treated with zVAD-fmk and/or Nec-1 prior to treatment with LPS. Cell viability was measured with the MTS assay (n = 3 mice). Error bars represent SEM. (***p < 0.001)
Fig. 2
Fig. 2
RIPK1 kinase inactive mice are protected from TNF- and TNF/zVAD-induced shock. Body temperature and survival of WT and Ripk1D138N/D138N mice injected with TNF (a) or TNF/zVAD (b). Error bars represent SEM. (***p < 0.001, ****p < 0.0001). (Originally published in The Journal of Immunology. Polykratis A, Hermance N, Zelic M et al. 2014. Cutting edge: RIPK1 kinase inactive mice are viable and protected from TNF-induced necroptosis in vivo. J Immunol. 193:1539–1543. Copyright 2014. The American Association of Immunologists, Inc.)
See this image and copyright information in PMC

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