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Studying necroptosis and non-apoptotic cell death

Related

  • Cell death resources
    • Apoptosis analysis guide
      • Autophagy overview

          Find out how to identify necroptosis, pyroptosis and ferroptosis.

          Distinguishing between different forms of non-apoptotic cell death can be challenging, especially as many share similar morphological features. However, the distinct regulatory pathways involved with each provide distinct protein markers that can be used for their detection.

          Whether you are looking at necroptosis, pyroptosis or ferroptosis, a combination of different approaches should be used. The study of non-apoptotic cell death should use both specific positive indicators of the cell death mode of interest, coupled with other cell viability assays and techniques to rule out apoptosis1.

          Contents

          • Necroptosis pathways and detection
          • Detecting pyroptosis
          • Detecting ferroptosis
          For more information, download the full Necroptosis and non-apoptotic cell death application guide

          ​Necroptosis pathways and detection

          Necroptosis is a programmed form of necrosis that is dependent on activation of receptor-interacting kinase 3 (RIPK3)2 and the mixed lineage kinase domain-like (MLKL) pseudokinase3. This form of cell death is morphologically distinct from apoptosis, involving membrane rupture and release of cytoplasmic contents.

          Necroptosis pathway

          Necroptosis is activated in response to death receptor activation, although some death receptor-independent pathways are also a trigger. In most contexts, necroptosis is inhibited by proapoptotic caspase 84–6; certain intracellular pathogens suppress apoptosis by inhibiting caspase 8, and necroptosis plays a role as a back-up to eliminate infected cells7. 

          The full necroptosis pathway can be found in our full Necroptosis analysis guide

          ​

          Figure 1. The death receptor-dependent pathway of necroptosis

          Although many proteins are involved in the necroptotic pathway, the most reliable method to detect necroptosis is by measuring the MLKL phosphorylation status and by specific inhibition of the necroptotic pathway.

          Key proteins in the necroptotic pathway


          ProteinRole in necroptosis
          RIPK1Protein kinase that recruits RIPK3 to the necrosome, resulting in mutual phosphorylation of RIPK1 and RIPK3.
          RIPK3Protein kinase that phosphorylates MLKL8. Activated by phosphorylation by RIPK1 and subsequent oligomerization.
          MLKLKinase domain-like protein. Once phosphorylated by RIPK3, MLKL translocates to the cell membrane to mediate cell death9.
          Caspase-8Inhibits necroptosis10.

          MLKL phosphorylation

          MLKL is activated by RIPK3-mediated phosphorylation. The activation state of MLKL can be determined by measuring the phosphorylation status of Thr357 and Ser358. Phospho-MLKL is detected by antibody-based methods, including western blot, IHC, and flow cytometry.

          ​​​​​​


          Product highlight

          Anti-MLKL (phospho S358) antibody  (ab187091)

          Description: rabbit monoclonal

          Application: IHC-P, WB

          Reactivity: human

          Image: staining human skin at 1/250 by IHC (FFPE)


          Necroptosis inhibition

          Targeting components of the necroptotic pathway – either by chemical inhibition or with transgenic models – can be used to tell whether cell death is dependent on necroptosis. 

          CompoundTarget
          Necrostatin-1 (Nec1)RIPK1
          7-Cl-O-Nec-1 (Nec1s)RIPK1
          GSK'872RIPK3
          NecrosulfonamideMLKL

          Considerations when using inhibitors:

          • Nec 1 has some off-target activity; Nec1s is more specific11.
          • RIPK1 can contribute to apoptosis12. Be aware that RIPK1 inhibitors may also block apoptosis under some circumstances.
          • Using transgenic models is the best method for confirming the presence of necroptosis.

          Detecting pyroptosis

          Pyroptosis is an inflammatory caspase-dependent form of programmed necrosis that occurs in response to microbial infection. Morphologically, pyroptotic cells display cell swelling and rapid plasma membrane lysis. Pyroptosis can be studied by looking at caspase activation, gasdermin D cleavage, or by inhibiting or ablating key components of the pyroptotic pathway.

          Key components of the pyroptotic pathway

          ProteinFunctionRole in pyroptosis
          Caspase 1Inflammatory caspase, activated by sensor proteins and inflammatory agentsCleaves gasdermin D
          Caspase 11 (mouse) or
          Caspase 4 and 5 (human)
          Inflammatory caspases, activated by bacterial polysaccharidesCleaves gasdermin D
          Gasdermin DCleaved by caspases13–15Executes pyroptosis


          Caspase activity

          Active caspases are cleaved from their inactive pro-caspase forms during pyroptosis. Caspase cleavage can be detected by western blot, using a specific caspase antibody.
          ​


          Product highlight

          Anti-caspase 11 rabbit monoclonal antibody (ab180673)

          Description: rabbit monoclonal

          Applications: western blot, IHC

          Reactivity: mouse

          Image: Antibody at 1/1000 dilution. Lane 1, untreated RAW 164.7 cell lysate. Lane 2, RAW 146.7 cells treated with lipopolysaccharide.

          Although active caspases are cleaved, observing caspase cleavage alone is not proof of caspase activation, and other methods should also be used to confirm pyroptosis. Caspase activation can be detected directly using caspase activation assays.



          Product highlight

          Caspase 1 assay kit (fluorometric, ab39412)

          Sample type: tissue extract, cell lysate

          Assay time: 2 hours

          Image: titration of caspase 1, background subtracted


          Gasdermin D

          Pyroptosis involves cleavage of gasdermin D (53 kDa), resulting in a 30 kDa N-terminal fragment, detected by western blot.

          We recommend using our anti-gasdermin D rabbit polyclonal (ab155233), which detects the N-terminal region of gasdermin D.

          Pyroptosis inhibition

          Showing dependence on caspase 1, 11, 4 or 5 is essential to distinguish pyroptotic cell death from other forms of necroptosis and apoptosis. Determine if cell death still occurs after ablation of these caspases, either by chemical inhibition or using transgenic models.

          Caspase 1 activity can be ablated by chemical inhibition with z-YVAD-fmk (ab141388).

          Detecting ferroptosis

          Ferroptosis is an iron-dependent form of cell death that occurs as a consequence of lipid reactive oxygen species (ROS) production16. Cells undergoing ferroptosis exhibit subtle morphological features, including smaller-than-normal mitochondria with increased density. The presence of ferroptosis can be confirmed by looking at whether cell death is prevented by inhibitors, and by measuring lipid peroxides.

          Key proteins in the ferroptotic pathway

          ProteinFunctionRole in ferroptosis
          GPX4Reduces lipid hydroperoxides within lipid membranesActivity reduced in ferroptosis
          GlutathioneSubstrate for GPX4Sometimes depleted in ferroptosis, depending on the molecular pathway


          Inhibiting ferroptosis

          The presence of ferroptosis can be confirmed using chemical inhibitors known to prevent ferroptosis. As ferroptosis is caused by reduction of GPX4 activity, knockdown is not an effective method.

          Ferroptosis inhibitors and their modes of action:

          InhibitorMode of action
          Ferrostatin-1Lipid peroxide scavenger
          Liproxstatin-1Unknown. Possibly reduction of free radicals



          Accumulation of lipid peroxides

          Ferroptosis is dependent on lipid reactive oxygen species (ROS) accumulation. A number of methods are available to detect the presence of lipid ROS.

          Methods to detect the presence of lipid ROS

          AssayMechanismHow to measure
          C11-BODIPYDetects free radical-induced oxidationQuantification by flow cytometry
          Malondialdehyde quantificationMalondialdehyde is a biproduct of lipid peroxidationLipid peroxidation (MDA) assay kit
          4-HNE quantification4-HNE is a biproduct of lipid peroxidationAntibody-based quantification



          Product highlight

          Glutathione peroxidase assay kit (colorimetric, ab102530)

          Sample type: cell culture supernatant, urine, serum, plasma, platelets, tissue extracts

          Sensitivity: 0.5 mU/mL

          Image: Glutathione peroxidase activity measured in cell lysates. Data are per million cells after a 10-minute incubation

          ​​

          ​​

          ​References

          1. Vanden Berghe, T. et al. Determination of apoptotic and necrotic cell death in vitro and in vivo. Methods 61, 117–129 (2013).

          2. Moriwaki, K. & Chan, F. K. M. RIP3: A molecular switch for necrosis and inflammation. Genes Dev. 27, 1640–1649 (2013).

          3. Sun, L. et al. Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell 148, 213–227 (2012).

          4. Donnell, M. A. O. et al. NIH Public Access. 13, 1437–1442 (2012).

          5. Lin, Y., Devin, A., Rodriguez, Y. & Liu, Z. G. Cleavage of the death domain kinase RIP by Caspase-8 prompts TNF-induced apoptosis. Genes Dev 13, 2514–2526 (1999).

          6. Feng, S. et al. Cleavage of RIP3 inactivates its caspase-independent apoptosis pathway by removal of kinase domain. Cell Signal 19, 2056–2067 (2007).

          7. Mocarski, E. S., Guo, H. & Kaiser, W. J. Necroptosis: The Trojan horse in cell autonomous antiviral host defense. Virology 479–480, 160–166 (2015).

          8. Sun, L. et al. Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell 148, 213–227 (2012).

          9. Dixon, S. J. et al. Ferroptosis: an iron-dependent form on nonapoptotic cell death. 149, 1060–1072 (2012).

          10. Moriwaki, K. & Chan, F. K. M. RIP3: A molecular switch for necrosis and inflammation. 

          11. Takahashi, N. et al. Necrostatin-1 analogues: critical issues on the specificity, activity and in vivo use in experimental disease models. Cell Death Dis 3, e437–10 (2012).

          12. Kaiser, W. J. et al. RIP1 suppresses innate immune necrotic as well as apoptotic cell death during mammalian parturition. Proc Natl Acad Sci 111, 7753–7758 (2014).

          13. He, W. et al. Gasdermin D is an executor of pyroptosis and required for interleukin-1[beta] secretion. Cell Res 25, 1285–1298 (2015).

          14. Shi, J. et al. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526, 660–665 (2015).

          15. Kayagaki, N. et al. Caspase-11 cleaves gasdermin D for non-canonical inflammasome signaling. Nature 526, 666–671 (2015).

          16. Dixon, S. J. et al. Ferroptosis: an iron-dependent form on nonapoptotic cell death. 149, 1060–1072 (2012).



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