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Phospho-RIPK1 (Ser 166) Cellular Kit HTRF®

This HTRF kit enables the cell-based quantitative detection of phosphorylated RIPK1 at Ser166 as a readout of the programmed necrotic cell death pathway, also called necroptosis

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  • No-wash No-wash
  • High sensitivity High sensitivity
  • All inclusive kit All inclusive kit
  • Low sample consumption Low sample consumption

This HTRF kit enables the cell-based quantitative detection of phosphorylated RIPK1 at Ser166 as a readout of the programmed necrotic cell death pathway, also called necroptosis

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Overview

This HTRF cell-based assay conveniently and accurately detects phosphorylated RIPK1 at Ser166. ​​​​​​​

RIPK1 (Receptor-Interacting serine/threonine-Protein Kinase 1 , also called RIP1 or RIP) is a key mediator of apoptotic and necrotic cell death, as well as of inflammatory pathways downstream of TNFR1 and other receptors. RIPK1 autophosphorylation at Serine 166 is considered to be a biomarker of the initiation of RIPK3/MLKL-dependent necroptosis. 

Necroptosis is a programmed necrotic cell death pathway, also called "inflammatory cell death", which is closely associated with pathologies including inflammatory and neurodegenerative diseases, as well as cancer. RIPK1 has therefore become an important drug target in the pharmaceutical industry. Therapeutic strategies consist in developing specific small-molecule kinase inhibitors, such as the well-known Necrostatin-1 and Necrostatin-1s. 

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Phospho-RIPK1 (Ser166) assay principle

The Phospho-RIPK1 (Ser166) assay measures RIPK1 when phosphorylated at Ser166. 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.
Principle of the HTRF phospho-RIPK1 (Ser166) assay

Phospho-RIPK1 (Ser166) two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates to a 384-well low volume detection plate before the addition of Phospho-RIPK1 (Ser166) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Two-plate protocol of the HTRF phospho-RIPK1 (Ser166) assay

Phospho-RIPK1 (Ser166) one-plate assay protocol

Detection of Phosphorylated RIPK1 (Ser166) 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.

One-plate protocol of the HTRF phospho-RIPK1 (Ser166) assay

Modulation of RIPK1 kinase activity in RT-112/84 cells

The human bladder cancer cell line RT-112/84 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 RIPK1 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. For RIPK1 inhibition experiments, the cells were pre-treated with 25 µM Z-VAD for 20 minutes before the addition of increasing doses of Necrostatin-1s. After 10 additional minutes, a pre-mix containing 100 ng/mL TNF-α and 100 nM SM-164 was added for 6h. After treatment, the cells were lyzed with 50 µL of supplemented lysis buffer #1 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-RIPK1 (Ser166) or Total-RIPK1 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 RIPK1 autophosphorylation at Ser166, highlighting the activation of the necroptosis pathway. In accordance with the literature (1,2), the total RIPK1 level is inversely modulated due to the association of RIPK1 with other necroptotic partners ('complex IIb') and the formation of a highly insoluble structure that remains in the insoluble fraction of the cell lysate.

As expected, the RIPK1 inhibitor Necrostatin-1s prevents TNF-α-induced necroptosis initiation by blocking RIPK1 autophosphorylation.

For both experiments, the level of alpha-tubulin was also monitored. The results show unchanged levels of the housekeeping protein, demonstrating that the modulations observed on Phospho- and Total RIPK1 are induced by the pharmacological treatments, and are not caused by cell detachment that can happen during late stages of necroptosis.


(1) Zhu et al. Cell Death and Disease (2018) 9:500

(2) Lee et al. Cell Death and Disease (2019) 10:923

Activation of RIPK1 in RT-112/84 cells using TNF-alpha
Inhibition of RIPK1 in RT-112/84 cells using Necrostatin-1s

Prevention of necroptosis activation in HT-29 cells using RIPK1 inhibitors

The human colorectal cancer cell line HT-29 was seeded in a 96-well culture-treated plate under 100,000 cells/well in complete culture medium, and incubated overnight at 37 °C, 5% CO2. The cells were pre-treated with 25 µM Z-VAD for 20 minutes before the addition of increasing doses of Necrostatin-1s or Necrostatin-1. After 10 additional minutes, a pre-mix containing 100 ng/mL TNF-α and 100 nM SM-164 was added for 6h. After treatment, the cells were lyzed with 50 µL of supplemented lysis buffer #1 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-RIPK1 (Ser166) or Total-RIPK1 detection reagents were added. The HTRF signal was recorded after an overnight incubation.

The RIPK1 inhibitor Necrostatin-1 and its analog Necrostatin-1s both induce a dose-dependent decrease in RIPK1 autophosphorylation at Ser166, and inversely a dose-dependent increase in total RIPK1 in the soluble fraction of the cell lysate. As expected, Necrostatin-1s is more potent than the original small molecule, based on the IC50 and EC50 values obtained on Phospho- and Total RIPK1 that are ~ 3 times better than with Necrostatin-1.

Validation of Phospho-RIPK1 in human HT-29 cells using RIPK1 inhibitors
Validation of Total RIPK1 in human HT-29 cells using RIPK1 inhibitors

Inhibition of RIPK1 kinase activity in SH-SY5Y cells using Necrostatin-1s

The human neuroblastoma cell line SH-SY5Y 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. The cells were pre-treated with 25 µM Z-VAD for 20 minutes, before the addition of increasing doses of Necrostatin-1s. After 10 additional minutes, a pre-mix containing 100 ng/mL TNF-α and 100 nM SM-164 was added for 6h. After treatment, the cells were lyzed with 25 µL of supplemented lysis buffer #1 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-RIPK1 (Ser166) or Total-RIPK1 detection reagents were added. The HTRF signal was recorded after an overnight incubation.

Necrostatin-1s is also able to prevent the initiation of the necroptosis pathway in neuronal cells, as indicated by the inhibition of RIPK1 autophosphorylation at Ser166. In this cell line model, no modulation of Total RIPK1 was observed.

Inhibition of RIPK1 in SH-SY5Y cells using Necrostatin-1s

HTRF Phospho-RIPK1 (S166) 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 #1 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-RIPK1 (Ser166) detection reagents. Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.

Using the HTRF phospho-RIPK1 (Ser166) assay, 12,500 cells/well were enough to detect a significant signal, while 25,000 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore, in these conditions, the HTRF phospho-RIPK1 assay was 2 times more sensitive than the Western Blot technique.

Comparison between HTRF and WB on Phospho-RIPK1 (S166)

RIPK1 Signaling Pathway

RIPK1 is a master regulator of cell fate-decisions by governing cell survival, apoptosis, and necroptosis pathways.

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, RIPK1 interacts with RIPK3 and MLKL to form the cytosolic complex IIb (also called 'necrosome') which is involved in the initiation of necroptosis. The activation of RIPK1 by autophosphorylation at Ser166 is essential to this complex and triggers RIPK3 and MLKL activation by phosphorylation. MLKL is the most downstream effector of necroptosis: the protein oligomerizes and translocates to the plasma membrane, leading to cell membrane rupture and the release of DAMPs.

RIPK1 signaling pathway

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