Studying non-apoptotic cell death
Related
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.
Whichever mode of cell death you are studying, a combination of different approaches should be used. 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
Detecting necroptosis
Necroptosis is initiated by a ligand binding to death receptors, including tumor necrosis factor receptor 1 (TNFR1)2. 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 MLKL3. 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 death11. |
| Caspase-8 | Inhibits necroptosis5. |
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.
| 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 specific6.
- RIPK1 can contribute to apoptosis7. 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 caspases8–10 | Executes pyroptosis |
Caspase activity
Active caspases are cleaved from their inactive pro-caspase forms during pyroptosis. Caspase cleavage can de detected by western blot, using a specific caspase antibody.
| |
| 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.
| |
| 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) production11. 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 |
| |
| 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.Holler, N. et al. Fas triggers an alternative, caspase-8–independent cell death pathway using the kinase RIP as effector molecule. Nat Immunol 1, 489–495 (2000).
- 3. Sun, L. et al. Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell 148, 213–227 (2012).
- 4. Cai, Z. et al. Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis. Nat Cell Biol 16, 55–65 (2014).
- 5. Moriwaki, K. & Chan, F. K. M. RIP3: A molecular switch for necrosis and inflammation. Genes Dev 27, 1640–1649 (2013).
- 6. 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).
- 7. 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).
- 8. He, W. et al. Gasdermin D is an executor of pyroptosis and required for interleukin-1[beta] secretion. Cell Res 25, 1285–1298 (2015).
- 9. Shi, J. et al. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526, 660–665 (2015).
- 10. Kayagaki, N. et al. Caspase-11 cleaves gasdermin D for non-canonical inflammasome signaling. Nature 526, 666–671 (2015).
- 11.Dixon, S. J. et al. Ferroptosis: an iron-dependent form on nonapoptotic cell death. 149, 1060–1072 (2012).