AlphaLISA SureFire Biotin-Free Human Total STING Detection Kit, 100 Assay Points

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

After treatment, the cells were spun down 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 Biotin Free 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, peaking at 4 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 (clone E9A9K, #72650).

THP-1 cells were seeded in a 96-well plate (400,000 cells/mL) in complete medium and transfected with 200 µg/mL of 2’3’cGAMP at the indicated time points.

After treatment, the cells were spun down 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 Biotin Free 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, 2’3’cGAMP induced STING phosphorylation, peaking at 3-4 hours with a modest decrease in STING total levels.

Dose-dependent activation of STING phosphorylation

THP-1 cells were seeded in a 96-well plate (400,000 cells/well) in complete medium and transfected with 20 µg/mL Poly dA/dT at the indicated concentrations for 4 hours.

After treatment, the cells were spun down and lysed with 100µL 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 Biotin Free assays. For the detection step, 10 µL of diluted 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, Poly dA/dT triggered a dose-dependent increase in the levels of Phospho STING (Ser366) while Total levels remained unchanged.

STING expression in various cell lines

Adherent cells were seeded in a 96-well plates (40,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. Cells were lysed with 100 µL Lysis buffer. Suspension cells were harvested and seeded in a 96-well plate (400,000 cells/well). Cells were lysed with 100µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STING levels were evaluated using the AlphaLISA SureFire Ultra Biotin Free assay. For the detection step, 10 µL of cell lysate (4,000 adherent cells or 40,000 suspension cells) were 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.

STING expression was detected in a wide range of human cell lines with higher levels observed in THP-1 and U937 cells.

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from THP-1 cells treated with 20 µg/mL of Calyculin A for 2 hours and lysed at 4 x 10⁶ cells/mL with Lysis Buffer B for 30 minutes at RT with shaking. Lysate was serially diluted and Phospho (Ser366) and Total STING levels were evaluated using the AlphaLISA SureFire Biotin Free assay. For the detection step, 10 µL of lysate was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. 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.

Approximate number of cells per datapoint is indicated. Dotted line represents assay background. This assay can detect STING expression in less than 500 cells/datapoint.