AlphaLISA SureFire Ultra Human & Mouse Phospho-STAT1 (Tyr701) Detection Kit, 500 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.

Induction of STAT1 (Tyr701) phosphorylation in primary macrophages treated with various cytokines

PBMCs were isolated from healthy donors and cultured for 6 days in complete DMEM containing 20 ng/mL M-CSF to differentiate them into macrophages. Macrophages were seeded in a 96-well plate (40,000 cells/well) in complete DMEM, and incubated overnight at 37°C, 5% CO2. Cells were starved for 2 h  and then treated with the indicated cytokines for 15 minutes.

After treatment, cells were lysed in 50 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT1 Phospho (Tyr701) levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 8,000 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.  

As expected, IFN type I and II were the main activators of STAT1 phosphorylation in primary macrophages.  

THP-1 cells were seeded in a 96-well plate (100,000 cells/well) in complete medium containing 100 nM of PMA for 24 hours at 37°C, 5% CO2. The THP-1 derived macrophages were starved for 2 hours in HBSS + 0.1% BSA and then treated with increasing concentrations of IFNγ for 20 minutes.

After treatment, the cells were lysed with 60 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT1 Phospho (Tyr701) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 16,000 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, IFNγ triggered a dose-dependent increase in the levels of Phospho STAT1 (Tyr701) while Total STAT1 levels remained unchanged. 

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 mouse IFNγ for 20 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT1 Phospho (Tyr701) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 4,000 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, IFNγ triggered a dose-dependent increase in the levels of Phospho STAT1 (Tyr701) while Total STAT1 levels remained unchanged.