AlphaLISA SureFire Ultra Human & Mouse Phospho-STAT3 (Tyr705 ) 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 STAT3 (Tyr705) phosphorylation in primary cells treated with various cytokines

PBMCs were isolated from healthy donors using Ficoll Plaque Plus (Merck, GE17-1440-02), Cells were seeded in a 96-well plate (400,000 cells/well) and starved for 2 hours in DMEM containing 1% FBS. Cells were then treated with the indicated cytokines for 15 minutes.

After treatment, cells were lysed in 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT3 Phospho (Tyr705) 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.  

As expected, IL-6 and IFN type I and II were the main activators of STAT3 phosphorylation in PBMCs

Activation of Phospho STAT3 (Tyr705) in primary macrophages

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 (30,000 cells/well) in complete DMEM, and incubated overnight at 37°C, 5% CO2.  The cells were then treated with increasing concentrations of IFNa for 10 minutes.

After treatment, the cells were lysed with 150 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT3 Phospho and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays.

For the detection step, 10 µL of cell lysate (approximately 2,600 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, IFNα triggered a dose-dependent increase in the levels of Phospho STAT3 (Tyr705) while Total STAT3 levels remained unchanged.

Induction of Phospho STAT3 (Tyr705) in IFNα treated cells

THP-1 cells were seeded in a 96-well plate (100,000 cells/well) in RPMI 1640 complete medium containing 100 nM PMA and incubated for 24 hours at 37°C, 5% CO2. The THP-1 derived macrophages were washed and treated with increasing concentrations of IFNα for 10 minutes in HBSS + 0.1% BSA.

After treatment, the cells were lysed with 50 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). STAT3 Phospho (Tyr705) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. 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.

As expected, IFNa triggered a dose-dependent increase in the levels of Phospho STAT1 (Tyr705) while Total STAT1 levels remained unchanged.