Tips for investigating cell death (Part 2) – Necrosis, necroptosis, pyroptosis  

Wanted dead or alive – Tips for investigating cell death (Part 2)

– Necrosis, necroptosis, pyroptosis 

The second part of the post series introduces three pro-inflammatory forms of cell death: necrosis, necroptosis and pyroptosis. The first part of the article series introduced autophagy and apoptosis are controlled cell events, that don’t cause an inflammatory response, necrosis, necroptosis and pyroptosis induce inflammation. Keep reading to learn how the cell death modes can be activated and detected!  

Necrosis 

Necrosis is an unregulated form of cell death, which can be caused by infection, hypoxia or injury – an outside stimuli of damage. In necrosis the cell membrane integrity is lost and eventually lysed, cell organelles swell and ATP, DNA and nuclear proteins are released, which will cause an inflammatory response. (1) 

Activation: Necrosis can be activated by culturing cells in hypoxic conditions, repeating cold/heat cycles 

Tips for detecting necrosis: Under the microscope necrosis can be detected by looking to morphology of the cell, swelling of cell and organelles. The most common method for measuring necrosis is lactate dehydrogenase LDH assay. LDH is a soluble cytoplasmic enzyme that is released when cell membrane disintegrates (2). 

#601170 LDH Cytotoxicity Assay Kit  

Necroptosis 

Necroptosis is triggered by the same receptors as apoptosis but has a morphology of necrosis. It can be activated via death receptors like FAS and tumor necrosis factor receptor 1 (TNFR1). The signals can be recognized intra- or extracellularly. Commonly necroptosis happens in response to same stimuli as apoptosis and in cell culture conditions necroptosis can be achieved by using caspase inhibitors that prevent apoptosis or under low ATP levels. Necroptosis can be distinguished by release of interleukin 1β. (3) 

Activation: In HT-29 or L929 cells, induction with TNFα (AG-40B-0006, TNF-α, Soluble (human) (rec.) (AdipoGen) and simultaneously using SMAC mimetics/CHX (#14126 Cycloheximide, Cayman Chemicals), and Z-VAD-FMK (#27421 Z-VA-DL-D(OMe)-FMK (trifluoroacetate salt), Cayman Chemicals (4) 

Necroptosis inhibitors: #10528 Necrostatin-7 (Cayman chemicals), #11657 Necrostatin-2 (Cayman chemicals), #11658 Necrostatin-1 (Cayman chemicals)  

Tips for detecting necroptosis: 

IL-1β ELISA (human) (#AG-45B-0021-KI01 AdipoGen), IL-1β ELISA (mouse) (#KOA0211 Rockland), #601170 LDH Cytotoxicity Assay Kit 

#98110 Necroptosis Antibody sampler kit (Cell Signaling) 

Pyroptosis 

Pyroptosis is an inflammatory form of cell death where the inflammasome plays a major role and plasma membrane integrity is lost. It is a specific cell death type of monocytic cells (5). Inflammasome is activated by inflammasome sensors like Nod-like receptor (NLR) family, the DNA receptor Absent in Melanoma 2 (AIM2) and the Pyrin receptor. When the receptors are activated, they oligomerize and recruit the adapter ASCs to form a complex called inflammasome. This will lead to activation of caspases and eventually to Gasdermin D activation. Gasdermin D will form a pore complex on cell membrane enabling the release of mature Interleukin 1β and interleukin 18. The difference between pyroptosis and necroptosis is that even though the same cytokines are released, the plasma membrane doesn’t rupture in pyroptosis even though swelling does occur. (6) 

Activation: LPS (#23608 LPS from Salmonella minnesota R595 (Re)) and nigericin (#11437) on bone marrow derived macrophages. See the protocol in references (7) 

Tips for detecting pyroptosis: IL-1β ELISA (human) (#AG-45B-0021-KI01 AdipoGen), IL-1β ELISA (mouse) (#KOA0211 Rockland), Annexin V-FITC early apoptosis detection kit (flow cytometry) #6592 (Cell Signaling Technology), TUNEL kits (IF, F) different fluorophores (#25879 488 nm, #64936 640 nm and #48513 594 nm) 

Download a pathway map including the cellular pathways in necrosis, necroptosis and pyroptosis. 

References: 

1. Khalid N, Azimpouran M. Necrosis. [Updated 2023 Mar 6]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan-. Available from: 
2. Chan FK, Moriwaki K, De Rosa MJ. Detection of necrosis by release of lactate dehydrogenase activity. Methods Mol Biol. 2013;979:65-70. doi: 10.1007/978-1-62703-290-2_7. PMID: 23397389; PMCID: PMC3763497. 
3. Nikoletopoulou V, Markaki M, Palikaras K, Tavernarakis N. Crosstalk between apoptosis, necrosis and autophagy. Biochim Biophys Acta. 2013 Dec;1833(12):3448-3459. doi: 10.1016/j.bbamcr.2013.06.001. Epub 2013 Jun 13. PMID: 23770045. 
4. Chesnokov M, Khan I, Chefetz I. Induction and Detection of Necroptotic Cell Death in Mammalian Cell Culture. Methods Mol Biol. 2021;2255:119-134. doi: 10.1007/978-1-0716-1162-3_11. PMID: 34033099. 
5. Yu, P., Zhang, X., Liu, N. et al. Pyroptosis: mechanisms and diseases. Sig Transduct Target Ther 6, 128 (2021). https://doi.org/10.1038/s41392-021-00507-5 
6. Bertheloot D, Latz E, Franklin BS. Necroptosis, pyroptosis and apoptosis: an intricate game of cell death. Cell Mol Immunol. 2021 May;18(5):1106-1121. doi: 10.1038/s41423-020-00630-3. Epub 2021 Mar 30. PMID: 33785842; PMCID: PMC8008022.  
7. den Hartigh AB, Fink SL. Pyroptosis Induction and Detection. Curr Protoc Immunol. 2018 Aug;122(1):e52. doi: 10.1002/cpim.52. Epub 2018 Jul 20. PMID: 30028908; PMCID: PMC6339837. 

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