Phospho-MLKL (Ser358) assay principle

The Phospho-MLKL (Ser358) assay measures MLKL when phosphorylated at Ser358. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses 2 antibodies, one labeled with a donor fluorophore and the other with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independently of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies, and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein's phosphorylation state under a no-wash assay format.

Phospho-MLKL (Ser358) two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of Phospho-MLKL (Ser358) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospho-MLKL (Ser358) one-plate assay protocol

Detection of Phosphorylated MLKL (Ser358) with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

MLKL activation in TNFa stimulated HT-29 cells

The human colorectal cancer cell line HT-29 was seeded in a 96-well culture-treated plate under 200,000 cells/well in complete culture medium, and incubated overnight at 37 °C, 5% CO2. For MLKL activation experiments, the cells were pre-treated with 25 µM Z-VAD (pan-caspase inhibitor) for 30 minutes before the simultaneous addition of SM-164 (cIAP1/2 inhibitor) at 100 nM and human TNF-α at various concentrations for 6 hours. After treatment, the cells were lyzed with 50 µL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking. For the detection step, 16 µL of cell lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Phospho-MLKL (Ser358) or Total-MLKL detection reagents were added. The HTRF signal was recorded after an overnight incubation.

In presence of SM-164 and Z-VAD, that block cell survival and apoptosis respectively, TNF-α induces a dose-dependent increase in MLKL phosphorylation at Ser358, highlighting the activation of the necroptosis pathway. Total MLKL show unchanged levels of signal, demonstrating that the modulations observed on Phospho-MLKL is induced by the pharmacological treatments, and is not caused by cell detachment that can happen during late stages of necroptosis.

HTRF Phospho-MLKL (S358) assay compared to Western Blot

The human colorectal cancer cell line HT-29 was cultured in a T175 flask in complete culture medium for 48h at 37°C, 5% CO2. The cells were pre-treated with 25 µM Z-VAD for 30 minutes before the simultaneous addition of SM-164 at 100 nM and human TNF-α at 100 ng/mL for 6 hours. After treatment, the cells were lyzed with 3 mL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF phospho-MLKL (Ser166) detection reagents. Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.

Using the HTRF phospho-MLKL (Ser358) assay, 8,125 cells/well were enough to detect a significant signal, while 65,000 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore, in these conditions, the HTRF phospho-MLKL assay was 8 times more sensitive than the Western Blot technique.

MLKL signaling pathway

MLKL plays key role in TNF-a induced necroptosis, programmed cell death process.

Upon binding of TNF-α to TNFR1, the receptor triggers the rapid formation of complex I at the cytoplasmic membrane by recruiting multiple proteins, including RIPK1, TRADD, TRAF2, and the cellular inhibitors of apoptosis 1 and 2 (cIAP1/2). cIAP1/2 catalyze the polyubiquitination of RIPK1, leading to the recruitment of IKKα and IKKβ and the activation of the NF-κB pro-survival pathway.

In the absence of cIAP1/2, RIPK1 is released from TNFR1 and associates with FADD and caspase 8 to form the cytosolic complex IIa, responsible for caspase 8-dependent cell apoptosis.

When caspase 8 activity is inhibited or under pathological conditions, MLKL interacts with RIPK1 and RIPK3 to form the cytosolic complex IIb (also called 'necrosome') which is involved in the initiation of necroptosis. Following RIPK1 autophosphorylation MLKL is phosphorylated by RIPK3 at Ser358 leading to its oligomerization, localization to the plasma membrane and execution of programmed necrosis characterized by calcium influx and plasma membrane damage . DAMPs are then released to promote damage repairs.