Is there an established protein marker for necrosis (but not necroptosis) or perhaps certain assays that should be done to directly determine necrotic cell death?
Cyclophilin A is released from necroptotic cells as a result of cytoplasmic membrane permeabilization.
CypA release from necrotic cells may play a role in the effects of necrosis on surrounding tissue. Necrosis is typically associated with inflammation. Necroptosis occurs in models of neuronal ischemia and traumatic brain injury. Treatment with Nec-1 significantly reduces cell death and inhibits the recruitment of neutrophils to the injured area. Extracellular CypA has been shown to induce a rapid inflammatory response upon injection in vivo and to recruit neutrophils. This recruitment is dependent on the CypA receptor, CD147 (EMMPRIN). Signaling of CypA through CD147 is proposed to contribute to the migration of neutrophils into the joints in rheumatoid arthritis and possibly in the destruction of bone and cartilage in the disease.
[Reference]
Cell Death Differ. 2010 Dec; 17(12): 1942–1943.
Cyclophilin A release as a biomarker of necrotic cell death
Unfortunately, although the title of the paper says that Cyclophilin A is a marker for necrosis, the death that they induce and talk about it necroptosis. I could not find any marker that is specific for necrosis. As long as your caspases aren't inhibited (with the addition of pan caspase inhibitors like zVAD) and you don't see expression of apoptosis associated proteins, the manner of cell death is assumed to be necrosis. As far as I know, there is no specific marker protein for necrosis, you basically reach that conclusion by elimination (of other forms of cell death).
I agree with Julie, that there is no specific westernblot marker for differentiating between necrosis and necroptosis. But if we break down the necroptosis process, then the points where it differs from necrosis are
1. It is induced by death receptor subsets from the TNF super family
2. Formation of necrosome: excessive RIPK1/RIPK3 complex
3. Absence of Caspase
So basically it is RIPK1 and RIPK3 over expression complex that results in necroptosis. Hence, by detecting this complex on western, with proper controls, you should be able to differentiate between necrosis and necroptosis. Hopefully, this works or atleast helps you to deisgn an experiment.
Regards
Sushree
Just to follow up, you do not need to detect necrosome complex formation. If you see phosphorylation of RIP1, RIP3 and/or MLKL, this is confirmation enough for necroptosis. See the paper below for details.
http://www.pnas.org/content/111/31/E3206
To summarize, you rule out all other forms of cell death and thereby the only one left is necrosis.
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