AlphaLISA SureFire Ultra Human Phospho-STING (Ser366) Detection Kit, 500 Assay Points

Phospho-AlphaLISA SureFire Ultra two-plate assay protocol

The two-plate protocol involves culturing and treating the cells in a 96-well plate before lysis, then transferring lysates into a 384-well OptiPlate™ plate before the addition of Phospho-AlphaLISA SureFire Ultra detection reagents. This protocol permits the cells viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.

Phospho-AlphaLISA SureFire Ultra one-plate assay protocol

Detection of Phosphorylated target protein with AlphaLISA SureFire Ultra reagents can be performed in a single plate used for culturing, treatment, and lysis. No washing steps are required. This HTS designed protocol allows for miniaturization while maintaining AlphaLISA SureFire Ultra quality.

THP-1 cells were seeded in a 96-well plate (400,000 cells/well) in complete medium and transfected with 5 µg/mL Poly dA/dT at the indicated time points.

After treatment, the cells were washed with HBSS and lysed with 100 µL of Lysis Buffer B for 30 minutes at RT with shaking (350 rpm). STING Phospho (Ser366)* and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 40,000 cells or 4,000 cells for Total STING) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

As expected, Poly dA/dT induced STING phosphorylation peaking at 6-8 hours with no significant changes to STING total levels.

*The highly specific and sensitive Phospho STING monoclonal antibody used in this assay was developed by Cell Signaling Technology (#72650).

THP-1 cells were seeded in a 96-well plate (400,000 cells/well) in complete medium and treated with 20 µg/mL diABZI at the indicated time points.

After treatment, the cells were spun down, washed with HBSS and lysed with 100 µL of Lysis Buffer B for 30 minutes at RT with shaking (350 rpm). STING Phospho (Ser366) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 40,000 cells or 4,000 cells for Total STING) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

diABZI induced an increase in Phospho STING (Ser366) levels, triggering its degradation 1 hour post treatment.

Poly dA/dT induces STING phosphorylation in a dose-dependent manner

THP-1 cells were seeded in a 96-well plate (400,000 cells/well) in complete medium and transfected with Poly dA/dT at the indicated concentrations for 4 hours.

After treatment, the cells were spun down, washed with HBSS and lysed with 100 µL of Lysis Buffer B for 30 minutes at RT with shaking (350 rpm). STING Phospho (Ser366) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 40,000 cells or 4,000 cells for Total STING) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

As expected, Poly dA/dT triggered a dose-dependent increase in the levels of Phospho STING (Ser366) while Total levels remained unchanged.

2'3' cGAMP induces STING phosphorylation in a dose-dependent manner

THP-1 cells were seeded in a 96-well plate (400,000 cells/well) in complete medium and stimulated with 2'3' cGAMP at the indicated concentrations for 4 hours.

After treatment, the cells were spun down, washed with HBSS and lysed with 100 µL of Lysis Buffer B for 30 minutes at RT with shaking (350 rpm). STING Phospho (Ser366) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 40,000 cells or 4,000 cells for Total STING) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.

As expected, 2'3' cGAMP triggered a dose-dependent increase in the levels of Phospho STING (Ser366). Total STING levels were largely unchanged with a slight decrease seen at the highest agonist concentration (i.e. 2-fold).