AlphaLISA SureFire Ultra High Performance Human and 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.

HeLa and A549 cells were seeded in a 96-well plate (20,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. Cells were starved for 20 hours and then stimulated with increasing concentrations of IFNα for 30 minutes.

After treatment, the cells were lysed with 100 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 High Performance STAT3 assays. For the detection step, 10 µL of cell lysate (approximately 4,000 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, IFNα treatment resulted in a dose dependent increase in STAT3 (Tyr705) phosphorylation, while STAT3 Total levels remained unchanged.

A549 and HeLa cells were seeded in a 96-well plate (20,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. Cells were starved for 20 hours and then stimulated with increasing concentrations of IL-6 for 15 minutes.

After treatment, the cells were lysed with 100 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 High Performance STAT3 assays. For the detection step, 10 µL of cell lysate (approximately 4,000 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, IL-6 treatment resulted in a dose dependent increase in STAT3 (Tyr705) phosphorylation, while STAT3 Total levels remained unchanged.

RAW 264.7 cells were seeded in a 96-well plate (20,000 cells/well) in complete medium and incubated overnight at 37°C, 5% CO₂. Cells were starved for 2 hours and then stimulated with increasing concentrations of IFNγ (mouse) for 20 minutes.

After treatment, the cells were lysed with 100 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 High Performance STAT3 assays. For the detection step, 10 µL of cell lysate (approximately 4,000 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, IFNγ treatment resulted in a dose dependent increase in STAT3 (Tyr705) phosphorylation, while Total levels remained unchanged.

Inhibition of STAT3 (Tyr705) phosphorylation in THP-1 derived macrophages

THP-1 cells were seeded in a 96-well plate (50,000 cells/well) in complete medium containing 100 nM PMA and incubated for 24 hours at 37°C, 5% CO₂. The THP-1 derived macrophages were treated with increasing concentrations of Ruxolitinib for 2 hours and then stimulated with 10 ng/mL 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). STAT3 Phospho (Tyr705) and Total levels were evaluated using respective AlphaLISA SureFire Ultra High Performance STAT3 assays. For the detection step, 10 µL of cell lysate (approximately 5,000 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 Ruxolitinib resulted in a dose-dependent decrease in the levels of Phospho STAT3 (Tyr705), while Total levels remained unchanged.

THP-1 cells were seeded in a 96-well plate (50,000 cells/well) in complete medium containing 100 nM PMA and incubated for 24 hours at 37°C, 5% CO₂. The THP-1 derived macrophages were starved in HBSS + 0.1% BSA for 2 hours and then stimulated with increasing concentrations of IFNα for 20 minutes.

After treatment, the cells were lysed with 100 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 STAT3 assays. For the detection step, 10 µL of cell lysate (approximately 5,000 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.

Cell lysate was prepared from A431 cells cultured to confluency in a T175 flask, treated with 2 µg/mL EGF for 10 minutes and lysed in 10 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and STAT3 Phospho (Tyr705) and Total levels were evaluated using respective AlphaLISA SureFire Ultra High Performance STAT3 assays. For the detection step, 10 µL of cell lysate 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.

Approximate number of cells is indicated. The dotted line represents assay background. The STAT3 High Performance assays have a broad dynamic range and can detect STAT3 expression in less than 50 cells/datapoint.