AlphaLISA SureFire Ultra Human Total IRF7 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.

THP-1 cells were seeded in a 96-well plate (200,000 cells/well) in complete medium and treated with increasing concentrations of IFNα or IFNβ for 6 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). Total IRF7 and Cofilin Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 20,000 cells for Total IRF7 and 400 cells for Total Cofilin) 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 a dose-dependent increase in the levels of Total IRF7 with no change to Cofilin Total.

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 increasing concentrations of IFNα or IFNβ for 24 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 Total and Cofilin Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 2,000 cells for IRF7 Total and 40 cells for Cofilin Total) 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 a dose-dependent increase in the levels of Total IRF7 with no significant changes to Cofilin Total.

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 a dose-dependent increase in the levels of Phospho (Ser477) and Total IRF7.

IRF7 activation mediated by STING agonists

THP-1 cells were seeded in a 96-well plate (400,000 cells/well) in complete medium and treated with 20 µM of STING agonist, diABZI at the indicated timepoints.

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 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 agonist, diABZI, triggered a gradual increase to the Total levels of IRF7 with peak increase occurring 24 hours post treatment.

IRF7 activation mediated by STING agonists

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

Expression of Total IRF7 in various cell lines

Adherent cell lines were seeded in a 96-well plate (40,000 cells/well) and incubated overnight at 37°C, 5% CO2. Cells were lysed with 100 µL of Lysis Buffer at RT with shaking (350 rpm).

Suspension cell lines were seeded in a 96-well plate (400,000 cells/well) in HBSS + 0.1% BSA and then lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm).

IRF7 levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of cell lysate (approximately 4,000 adherent cells and 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.

As expected, expression of Total IRF7 is low in most cell lines tested. Moderate levels of expression were detected in THP-1 and HT 29 cells.