Studying necroptosis and non-apoptotic cell death

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

​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 8^4–6; 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

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^{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

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

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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. 

Compound	Target
Necrostatin-1 (Nec1)	RIPK1
7-Cl-O-Nec-1 (Nec1s)	RIPK1
GSK'872	RIPK3
Necrosulfonamide	MLKL

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

Protein	Function	Role in pyroptosis
Caspase 1	Inflammatory caspase, activated by sensor proteins and inflammatory agents	Cleaves gasdermin D
Caspase 11 (mouse) or Caspase 4 and 5 (human)	Inflammatory caspases, activated by bacterial polysaccharides	Cleaves gasdermin D
Gasdermin D	Cleaved by caspases13–15	Executes 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.
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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

Protein	Function	Role in ferroptosis
GPX4	Reduces lipid hydroperoxides within lipid membranes	Activity reduced in ferroptosis
Glutathione	Substrate for GPX4	Sometimes 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:

Inhibitor	Mode of action
Ferrostatin-1	Lipid peroxide scavenger
Liproxstatin-1	Unknown. 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

Assay	Mechanism	How to measure
C11-BODIPY	Detects free radical-induced oxidation	Quantification by flow cytometry
Malondialdehyde quantification	Malondialdehyde is a biproduct of lipid peroxidation	Lipid peroxidation (MDA) assay kit
4-HNE quantification	4-HNE is a biproduct of lipid peroxidation	Antibody-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

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​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).