Cynomolgus TNF alpha kit
No-wash kit to quantify released Cynomolgus TNF alpha
The Total STING kit is designed to monitor the expression level of cellular STING and can be used as a normalization assay for the phospho-STING kit.
The Total STING cellular assay monitors total STING, and can be used as a normalization assay with our phospho-STING kit. This kit is compatible with the buffers from the phospho-STING kit, so the same lysate can be used for analyses of both the phosphorylated and the total protein populations.
Following pathogen infection and binding of dsDNA to the cytoplasmic sensor cGAS, STING protein is phosphorylated by TBK1, enabling its binding to IRF3 which induces IFNs type 1 production. The STING pathway is then switched off by STING degradation involving autophagy.
In immuno-oncology, activating the STING pathway has shown promising anti-tumor effects in pre-clinical models and thus represents a therapeutic strategy to treat human cancer.
The Total STING assay quantifies the expression level of STING in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total-STING assay uses two labeled antibodies, one coupled to a donor fluorophore and the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of STING 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-STING HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Detection of Total STING 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.
Human THP1-R232 cells (from Invivogen) were plated under 25µl in 96-well plates (400,000 cells/well) in complete culture medium. The cells were stimulated with increasing concentrations of 2’3’cGAMP (5µL for 4 hours). Cells were then lysed with the addition of 10 µL of supplemented lysis buffer #4 at 4X for 30 minutes at RT under gentle shaking. 16µL of lysate were transferred into a 384 low volume white microplate before the addition of 4 µL of the HTRF® phospho-STING or total STING detection antibodies. In parallel, Alpha-tubulin housekeeping protein was analyzed from 4µL of cell lysate, using the corresponding HTRF alpha-tubulin housekeeping kit. Finally, HTRF signals were recorded after an overnight incubation at RT.
As described elsewhere, 2’3’cGAMP induced a significant activation of the STING pathway, leading to a 6-fold increase in STING phosphorylation. The 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.
400,000 THP1-R232 cells (from Invivogen) were plated under 25 µL in 96-well plates in complete culture medium. The cells were stimulated with increasing concentrations of 2’3’cGAMP (5 µL for 4 hours). Cells were then lysed with the addition of 10 µL of supplemented lysis buffer #4 at 4X for 30 minutes at RT under gentle shaking. 16 µL of lysate were analyzed either by HTRF or Western Blot, in parallel.
Signal quantification by HTRF enabled accurate statistical analysis of compound effect, as shown on the graphs below, where 400 µM of cGAMP significantly decreased both total and phospho-STING (statistical method used: One way-Anova, P value <0.0001).
The human THP1-R232 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 2’3’cGAMP for 4 hours, 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 total STING detection antibodies. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.
Using the Total STING kit, 4,000 cells/well were enough to detect a significant signal with HTRF and Western Blot. Therefore, in our experiment the detection sensitivity of total STING protein is similar between the two methods.
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 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, to switch off IFNb production.
Detailed protocol and direct comparison with WB - Posters
A solution for phospho-protein analysis in metabolic disorders - Posters
Mastering the art of cell signaling assays optimization - Guides
HTRF and WB compatible guidelines - Technical Notes
Insider Tips for successful sample treatment - Technical Notes
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
Increased flexibility of phospho-assays - Application Notes
Valuable guidelines for efficiently analyzing and interpreting results - Application Notes
Tumor xenograft analysis: HTRF versus Western blot - Application Notes
One technology across all samples - Application Notes
A single technology for 2D cells, 3D cells, and xenograft models - Posters
Protocol for tumor xenograft analysis with HTRF - Technical Notes
In collaboration with Bayer - Scientific Presentations
Unmatched ease of use, sensitivity and specificity assays - Videos
Study a pathway of interest in PBMC and T cells - Application Notes
Analysis of a large panel of diverse biological samples and cellular models - Posters
Analyse of PI3K/AKT/mTor translational control pathway - Application Notes
PI3K/AKT/mTor translational control pathway - Posters
Physiologically relevant results fo fast flowing research - Flyers
Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers
cGAS-STING signaling pathway from A to Z - Brochures
Featuring a panel of experts - Videos
Get the brochure about technology comparison. - Brochures
Clear overview of past, present and future of immunotherapy - Guides
A fun video introducing you to phosphorylation assays with HTRF - Videos
Data about HTRF IFNb correlation with gene reporter assay and ELISA - Application Notes
Complete autoimmunity review in a single White Paper - Guides
Unlock STING polymorphism with this valuable note - Application Notes
Check out our new guide featuring valuable insights on autoimmunity - Guides
Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.Let's find your reader