AlphaLISA SureFire Ultra Mouse Total STING Detection Kit, 10,000 Assay Points

Total-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 Total-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.

Total-AlphaLISA SureFire Ultra one-plate assay protocol

Detection of Total 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.

DMXAA-mediated STING phosphorylation

RAW 264.7 cells were seeded in a 96 well plate (60,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. The cells were treated with 20 µg/mL DMXAA mouse STING ligand for the indicated time points.

After treatment, the cells were lysed with 50 µL of Lysis Buffer B for 20 minutes at RT with shaking (350 rpm). STING Phospho (Ser365)* and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 12,000 cells) 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, DMXAA strongly induced STING phosphorylation leading to a 500-fold increase within an hour. A 2.7-fold decrease in total levels was observed around 4 hours post-treatment.

*The highly specific and sensitive Phospho STING monoclonal antibody used in this assay was developed by Cell Signaling Technology (clone D1C4T, #51865).

DMXAA induces STING phosphorylation in a dose-dependent manner

RAW 264.7 cells were seeded in a 96 well plate (40,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. The cells were treated with increasing concentrations of DMXAA mouse STING ligand for 1 hour.

After treatment, the cells were lysed with 50 µL of Lysis Buffer B for 20 minutes at RT with shaking (350 rpm). STING Phospho (Ser365) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 8,000 cells) 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, DMXAA induced STING phosphorylation in a dose-dependent manner while no significant changes were observed in total levels 1 hour post-treatment.

Poly dA/dT induces STING phosphorylation

RAW 264.7 cells were seeded in T25 flasks (2 x 10⁶ cells/mL) in complete medium and incubated overnight at 37°C, 5% CO₂. The cells were left untreated or transfected with 25 µg/mL Poly dA/dT for 6 hours.

After treatment, the cells were lysed with 1 mL of Lysis Buffer B for 20 minutes at RT with shaking (350 rpm). STING Phospho (Ser365) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 40,000 cells) 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 leading to a 17-fold increase while total levels did not change significantly.

2'3' cGAMP induces STING phosphorylation

RAW 264.7 cells were seeded in a 96 well plate (60,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. The cells were treated with 100 µg/mL 2'3' cGAMP for the indicated time points.

After treatment, the cells were lysed with 50 µL of Lysis Buffer B for 20 minutes at RT with shaking (350 rpm). STING Phospho (Ser365) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 12,000 cells) 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 an increase in the levels of mouse Phospho STING peaking at 2 hours. A significant decrease in Total STING levels was observed post-phosphorylation (4- to 11-fold), likely due to its degradation.

Expression of STING in various mouse cell lines

Adherent cell lines were seeded in a 96-well plate (60,000 cells/well) and incubated overnight at 37°C, 5% CO₂. Cells were lysed with 50 µL of Lysis Buffer B for 20 minutes at RT with shaking (350 rpm).

Suspension cell line (EL-4 cells) was seeded in a 96-well plate (200,000 cells/well) in HBSS + 0.1% BSA and then lysed with 100 µL of Lysis Buffer B for 10 minutes at RT with shaking (350 rpm).

STING Total levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 12,000 adherent cells or 20,000 suspension cells ) 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.