Table 2.
Evaluation of necroptosis
| Methods to Detect the Particular Necroptotic Signaling | Key Notes | References | |
|---|---|---|---|
| Specific molecular events strongly suggesting necroptosis being activated and/or executed | |||
| Activation of canonical RIP1-RIP3-MLKL axis through phosporylation | 1) Detection: immunostaining; 2) inhibitors of RIP1 (necrostatins, GSK2982772), RIP3 (GSK`872, GSK`843), and MLKL inhibitors (necrosulfonamide); 3) knockdown approaches | Phosphorylated RIP1 and RIP3 suggest necroptosis activation; however, phosphorylation of either residue of MLKL robustly identifies necroptotic environment | 42, 49–51, 64, 102, 105, 114, 146, 159, 165, 191, 203, 242, 244, 263 |
| MLKL phosphorylation and translocation to the plasma membrane | 1) Detection: immunostaining, subcellular fractionation; 2) inhibitors: MLKL inhibitors (necrosulfonamide) | Evidence of MLKL in the membrane fraction indicates its prior activation by the phosphorylation and execution of necroptosis | 27, 33, 59, 240, 242, 243, 263, 268 |
| MLKL oligomerization in the nucleus and plasma membrane | Immunofluorescence, nonreducing SDS-PAGE, native page gels | Phosphorylation of MLKL is a prerequisite for homo/hetero-oligomerization and translocation to the membrane/nucleus | 96, 242, 263 |
| Necrosome formation: RIP1-RIP3 interaction, RIP3-MLKL interaction | Immunofluorescence, coimmunoprecipitation | Necrosome identification indicates necroptosis activation; however, end-stage effector of the necroptosis (p-MLKL) should be also provided | 34, 139, 194, 268, 286 |
| Other more nonspecific characteristics of necroptosis | Methods to detect nonspecific features of necroptotic cell death | ||
| Loss of cellular integrity, plasma membrane rupture | LDH, HMGB1 release, staining with impermeant dyes, annexin V/PI staining | ||
| Altered cell viability | XTT, MTT assay, etc. | ||
HMGB1, high-mobility group box 1 protein; LDH, lactate dehydrogenase; MLKL, mixed-lineage kinase domain-like; p-MLKL, phosphorylated mixed-lineage kinase domain-like; RIP1 and -3, receptor-interacting protein kinase 1 and -3, respectively; XTT, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide.