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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.
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.|
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.
|Anti-MLKL (phospho S358) antibody (ab187091)|
Description: rabbit monoclonal
Application: IHC-P, WB
Image: staining human skin at 1/250 by IHC (FFPE)
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.
Considerations when using inhibitors:
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|
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
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
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.
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).
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|
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