AlphaLISA SureFire Ultra Human Total IRF9 Detection Kit, 500 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.

IRF9 activation mediated by IFNβ

RPMI 8226 cells were seeded in a 96-well plate (200,000 cells/well) in complete medium and treated with 50 ng/mL of IFNβ at the indicated timepoints.

After treatment, the cells were washed with HBSS and then lysed with 100 µL of Lysis Buffer B for 10 minutes at RT with shaking (350 rpm). IRF9 levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of cell lysate (approximately 20,000 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 IFNβ triggered a gradual increase to the Total levels of IRF9 with peak induction occurring 7 hours post treatment with no change to Total Cofilin levels (data not shown).

Decrease of IRF9 levels by Ruxolitinib

THP-1 cells were seeded in a 96-well plate (300,000 cells/well) in complete medium and treated with increasing concentrations of Ruxolitinib for 24 hours.

After treatment, the cells were washed with HBSS and lysed with 100 µL of Lysis Buffer B for 10 minutes at RT with shaking (350 rpm). Total IRF9 and ERK1/2 Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 30,000 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.

Inhibition of JAK1 signaling mediated by Ruxolitinib, resulted in a significant decrease of Total IRF9 levels after 24 hours.

Knockout validation of IRF9 Total assay

Total IRF9 levels were assessed in HeLa wild type (WT) and HeLa IRF9 knockout (KO) (Abcam ab266051) cell lines.  HeLa WT and KO cells were seeded in a 96-well plate (10,000 cells/well) and incubated overnight at 37°C, 5% CO₂. Cells were treated with increasing concentrations of IFNα for 24 hours.

After treatment, cells were lysed in 100 µL of Lysis Buffer B for 10 minutes at RT with shaking (350 rpm). IRF9 Total levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of lysate (approximately 1,000 cells) 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.

Total IRF9 was only detected in HeLa WT cells treated with IFNα, demonstrating assay specificity.

Total IRF9 expression 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).

IRF9 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, basal Total IRF9 expression is low in most cell lines tested. Moderate levels of expression were detected in THP-1 cells.