Total RIPK1 Cellular Kit HTRF®

The HTRF Total-RIPK1 assay quantifies the expression level of RIPK1 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total-RIPK1 assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of RIPK1 in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor, and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.

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 Total-RIPK1 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Detection of Total-RIPK1 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.

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 the 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

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.

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.

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 total RIPK1 detection reagents. Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.

Using the HTRF total RIPK1 assay, 3,120 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 total RIPK1 assay was 8 times more sensitive than the Western Blot technique.

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 therelease of DAMPs.