Western Blotting and Flow Cytometry for Evaluating Necroptosis of Cells (Review)

The paper covers the current mainstream methods used for detecting the intensity of cellular necroptosis, which is considered to be an alternative mode of cell death that shares some features of apoptosis and necrosis. In particular, we have focused on Western blotting and flow cytometry as the basic techniques for evaluating necroptosis. This programmed type of cell death is associated with the cell membrane rupture and the release of intracellular content promoting its pro-inflammatory properties. In this review, we have described the major triggers of necroptosis, its molecular mechanisms, the role of necroptosis in pathogenesis of inflammatory diseases, as well as basic approaches used in the detection of cellular necroptosis intensity. Nowadays Western blotting and flow cytometry are supposed to be the major techniques used for evaluating the rate of cellular necroptosis. The detection of necroptosis by Western blotting is based on the determination of phosphorylated forms of specific receptor-interacting serine/threonine-protein kinase 1 (RIPK3) and receptor-interacting serine/threonine-protein kinase 1 (RIPK1), as well as pseudokinase mixed lineage kinase domain-like protein, involved in the necroptotic cell death signaling. The enzymes mentioned above are phosphorylated and therefore activated only when necroptosis is triggered. In addition, mixed lineage kinase domain-like protein oligomerization detected by Western blotting is also supposed to be a sign of necroptosis. Flow cytometry allows determining cellular necroptosis using a standard annexin V / propidium iodide kit with the subsequent imaging flow cytometry. Moreover, another flow cytometric approach recently discovered is based on the use of anti-RIPK3 and anti-active caspase-3 fluorescently labeled antibodies. Necroptotic cells are characterized by RIPK3+/active caspase-3- phenotype, since caspase-3 is not involved in necroptosis but is activated during apoptosis. It is worth mentioning that the ability to analyze necroptosis in individual cellular subpopulation seems to be an obvious advantage of flow cytometry method compared with Western blotting. Conclusions.We strongly believe that the development of new methods for assessing the activity of cellular necroptosis both in vivo and in vitro and the improvement of the existing ones can improve the understanding of the role of this pro-inflammatory cell death mode in pathogenesis of various diseases.