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Mouse Phospho-STING (Ser365) Cellular Kit HTRF®

This HTRF kit enables the cell-based quantitative detection of mouse phosphorylated STING as a readout of the activation of the cGAS-STING pathway.

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  • All inclusive kit All inclusive kit
  • No-wash No-wash
  • High sensitivity High sensitivity

This HTRF kit enables the cell-based quantitative detection of mouse phosphorylated STING as a readout of the activation of the cGAS-STING pathway.

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Overview

This HTRF cell-based assay conveniently and accurately quantifies mouse phosphorylated STING at Ser365. Following pathogen infection and binding of dsDNA to the cytoplasmic sensor cGAS, STING protein is phosphorylated by TBK1. This enables its binding to IRF3, which induces IFNs type 1 production and other immune responses. The STING pathway is then switched off by STING degradation involving autophagy.

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Mouse Phospho-STING (Ser365) assay principle

The Mouse Phospho-STING (Ser365) assay measures mouse STING when phosphorylated at Ser365. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The  Mouse Phospho-STING (Ser365) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one 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 independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving the two labeled antibodies. This 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 Mouse phospho-365 STING assay principle

Mouse Phospho-STING (Ser365) two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates to a low volume detection plate (either HTRF 384-lv or 96-lv plate) before the addition of HTRF Mouse Phospho-STING (Ser365) detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Two-plate protocol of the HTRF Mouse phospho-365 STING assay

Mouse Phospho-STING (Ser365) one-plate assay protocol


Detection of Mouse  Phosphorylated STING (Ser365) 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 Mouse phospho-365 STING assay protocol

DMXAA induces Mouse STING phosphorylation in RAW 264.7 and NIH3T3 cells

Raw264.7  & NIH3T3 cells were plated under 50 µl in a 96-well plates (200,000 cells/well and 100,000 cells/well respectively) in complete culture medium and incubated overnight . The cells were stimulated with increasing concentrations of mouse specific ligand DMXAA  (50µL for 1 hour). After treatment, cells were lysed with 50µl of supplemented lysis buffer #4 (1X) for 30 min at RT under gentle shaking. 16 µL of lysate were transferred into a 384 low volume white microplate before adding 4 µL of the HTRF phospho-Mouse STING or total Mouse STING detection antibodies.  Finally, HTRF signals were recorded after 2 hours of incubation at RT. DMXAA induced a significant activation of the STING pathway, leading to a 8 and 6 -fold increase of STING phosphorylation in Raw264.7 and NIH3T3 cells respectively.

Validation of phospho & Total Mouse STING on RAW264.7
Validation of phospho & Total Mouse STING on NIH3T3

ADUS100 induces Mouse STING phosphorylation in RAW 264.7 and NIH3T3 cells

Raw264.7  & NIH3T3 cells were plated under 50 µl in a 96-well plates (200,000 cells/well and 100,000 cells/well respectively) in complete culture medium and incubated overnight . The cells were stimulated with increasing concentrations of c-di-AMP analog : ADU-S100 (50µL for 1 hour). After treatment, cells were lysed with 50µl of supplemented lysis buffer #4 (1X) for 30 min at RT under gentle shaking. 16 µL of lysate were transferred into a 384 low volume white microplate before adding 4 µL of the HTRF phospho-Mouse STING or total Mouse STING detection antibodies.  Finally, HTRF signals were recorded after 2 hours of incubation at RT. ADUS100 induced a significant activation of the STING pathway, leading to a 9 and 5 -fold increase of STING phosphorylation in Raw264.7 and NIH3T3 cells respectively. Induced STING phosphorylation was associated with a down-regulation of its expression level, in agreement with autophagy mediated degradation.

Validation of phospho & Total Mouse STING on RAW264.7
Validation of phospho & Total Mouse STING on RAW264.7 and NIH3T3 cells

3’3’cGAMP induces Mouse STING phosphorylation in Raw264.7 cells

Raw264.7 cells were plated under 50 µl in a 96-well plates (200,000 cells/well) in complete culture medium and incubated overnight . The cells were stimulated with increasing concentrations of bacterial cyclic di-nucleotide ligand : 3'3'cGAMP  (50µL for 1 hour). After treatment, cells were lysed with 50µl of supplemented lysis buffer #4 (1X) for 30 min at RT under gentle shaking. 16 µL of lysate were transferred into a 384 low volume white microplate before adding 4 µL of the HTRF phospho-Mouse STING or total Mouse STING detection antibodies.  Finally, HTRF signals were recorded after 2 hours of incubation at RT. 3'3'cGAMP induced a significant activation of the STING pathway, leading to a  5 -fold increase of STING phosphorylation.

Validation of phospho & Total Mouse STING on Raw264.7 cells

2’3’cGAMP induces Mouse STING phosphorylation in Raw264.7 cells

Raw264.7 cells were plated under 50 µl in a 96-well plates (200,000 cells/well) in complete culture medium and incubated overnight . The cells were stimulated with increasing concentrations of non-canonical cGAMP  : 2'3'cGAMP  (50µL for 1 hour). After treatment, cells were lysed with 50µl of supplemented lysis buffer #4 (1X) for 30 min at RT under gentle shaking. 16 µL of lysate were transferred into a 384 low volume white microplate before adding 4 µL of the HTRF phospho-Mouse STING or total Mouse STING detection antibodies.  Finally, HTRF signals were recorded after 2 hours of incubation at RT. 3'3'cGAMP induced a significant activation of the STING pathway, leading to a  4 -fold increase of STING phosphorylation. Induced STING phosphorylation was associated with a down-regulation of its expression level, in agreement with autophagy mediated degradation, whereas alpha-tubulin remained stable under the same conditions.

Validation of Phospho & Total Mouse STING on Raw 264.7 cells
Validation of Phospho & Total Mouse STING on Raw 264.7 cells

HTRF phospho- Mouse STING cellular assay compared to Western Blot

Raw264.7 cell line was seeded in a T175 flask in complete culture medium, and incubated for 2 days at 37°C, 5% CO2. Cells were then stimulated with 100 µM DMXAA for 1 hour and lysed with 3 mL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking. Soluble supernatants were collected after a 10-minute centrifugation.Serial dilutions of the cell lysate were performed in the 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- Mouse STING detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.

A side by side comparison of Western Blot and HTRF demonstrates that both technology give the same sensitivity under these experimental conditions.

Comparison of HTRF Mouse phospho S-365  STING kit with Western Blot

STING simplified pathway

STING, for STimulator of INterferon Genes, is a cytoplasmic homodimeric protein localized in the endoplasmic reticulum,  which plays an essential role in innate immunity. Upon pathogen infection or mitochrondrial shrinking during apoptosis, floating dsDNAs bind and activate a DNA sensor, the cyclic GMP-AMP synthase (cGAS). Activated cGAS leads to the production of 2’-3’cGAMP, a cyclic dinucleotide, which then binds to STING proteins. In turn, phosphorylated STING interacts with TANK-binding-kinase-I (TBK1) leading to the recruitment and activation of active interferon regulatory factor (IRF3) dimer. Nuclear translocation of the IRF3 dimer leads to the transcription of genes encoding IFN-α/β. In addition, the STING pathway controls NF-κB dependent inflammatory cytokine expression. As a negative feedback loop, the DNA-stimulated cGAS-STING-TBK1 pathway also triggers STING protein degradation through p62 SQSTM1 associated autophagy, switching off IFNβ production.

STING mouse signaling pathway

HTRF cellular phospho-protein assays

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Plate Reader Requirement

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