AlphaLISA SureFire Ultra Human Phospho-IRF7 (Ser477) Detection Kit, 10,000 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 (100,000 cells/well) in medium containing 100 nM PMA and incubated for 24 hours at 37°C, 5% CO2. THP-1 derived macrophages were then treated with 250 ng/mL of IFNα or IFNβ for a further 24 hours.

After treatment, the cells were washed with HBSS and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). IRF7 Phospho (Ser477) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 10,000 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, treatment with type I interferons triggered an increase in the levels of Phospho (Ser477) and Total IRF7.

HT 29 cells were seeded in a 96-well plate (20,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO2. The cells were treated with 100 ng/mL of IFNα or IFNβ for 24 hours.

After treatment, the cells were washed with HBSS and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). IRF7 Phospho (Ser477) levels were evaluated using the AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 2,000 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.

Treatment with type I interferons triggered the activation of Phospho (Ser477) IRF7 in HT 29 cells.

THP-1 cells were seeded in a 96-well plate (400,0000 cells/well) in complete medium and treated with 20 µM of STING agonist, diABZI at the indicated time points.

After treatment, the cells were washed with HBSS and then lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). IRF7 Phospho (Ser477) levels were evaluated using the 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.

Treatment with STING agonist, diABZI, triggered a significant increase in IRF7 phosphorylation from 30 minutes with peak activation at 45 minutes.

THP-1 cells were seeded in a 96-well plate (400,000 cells/well) in complete medium and treated with 100 µg/mL of STING ligand, 2’3’ cGAMP for 4 hours.

After treatment, the cells were washed with HBSS and then lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). IRF7 Phospho (Ser477) and Total levels were evaluated using the AlphaLISA SureFire Ultra assay. 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.

Treatment with STING ligand, 2’3’ cGAMP, triggered a 10-fold increase of Phospho IRF7 (Ser477) and only 2-fold increase for Total IRF7.

Activation of Phospho (Ser477) IRF7 in Calyculin A treated cells

THP-1 cells were seeded in a 96-well plate (100,000 cells/well) in medium containing 100 nM PMA and incubated for 18 hours at 37°C, 5% CO2. THP-1 derived macrophages were then treated with increasing concentrations of Calyculin A for 30 minutes.

After treatment, the cells were washed with HBSS and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). IRF7 Phospho (Ser477) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 10,000 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, treatment with Calyculin A triggered a dose-dependent increase in phosphorylated IRF7 (Ser477) with no change to IRF7 Total levels.