AlphaLISA SureFire Ultra Human and Mouse Phospho-FOXO1 (Ser256) 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.

Insulin induces FOXO1 phosphorylation in a dose-dependent manner

MCF7 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 starved for 2 hours and treated with increasing concentrations of insulin for 5 minutes prepared in serum free medium.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). FOXO1 Phospho (Ser256) and Total levels were evaluated using respective AlphaLISA SureFire Ultra 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, insulin triggered a dose-dependent increase in the levels of Phospho FOXO1 (Ser256) with no significant changes in Total levels.

Razuprotafib induces FOXO1 phosphorylation in HUVEC cells

HUVEC 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 starved for 2 hours and treated with increasing concentrations of Razuprotafib for 15 minutes.

After treatment, the cells were lysed with 25 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). FOXO1 Phospho (Ser256) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 12,000 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.

Razuprotafib treatment results in the activation of the Tie2 signaling pathway in endothelial cells and a dose-dependent increase in the levels of Phospho FOXO1 (Ser256) with no significant changes in Total levels.

Insulin growth factor induces FOXO1 phosphorylation in MCF7 cells

MCF7 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 starved for 2 hours and treated 500 ng/mL of IGF-1 for 5 minutes prepared in serum free medium.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). FOXO1 Phospho (Ser256) and Total levels were evaluated using respective AlphaLISA SureFire Ultra 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, IGF-1 increased the levels of Phospho FOXO1 (Ser256) with no significant changes in Total levels (data not shown).

Decrease in FOXO1 phosphorylation by PI3K inhibitor

HEK293 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 50 µM LY294002 for 24 hours.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). FOXO1 Phospho (Ser256) levels were evaluated using the AlphaLISA SureFire Ultra assay. 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, LY294002 treatment resulted in a decrease of phosphorylated FOXO1 at Ser256.

Knockout validation of Phospho FOXO1 (Ser256) assay

Phospho FOXO1 (Ser256) levels were assessed in HAP1 wild type (WT) and FOXO1 knockout (KO) cells. Cells were cultured to confluency in T175 flasks at 37°C, 5% CO_2. Cells were lysed in 4 mL of Lysis Buffer.

Lysates were serially diluted in Lysis Buffer and FOXO1 Phospho (Ser256) levels were evaluated using the AlphaLISA SureFire Ultra kit. For the detection step, 10 µL of lysate 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.

Phospho FOXO1 (Ser256) was only detected in WT cells, confirming assay specificity.

Assay sensitivity - cell lysate

Cell lysate was prepared from HEK293 cells cultured to confluency in a T175 flask and lysed in 3 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and assayed for Phospho (Ser256) and Total FOXO1 levels using respective AlphaLISA SureFire Ultra kits. For the detection step, 10 µL of lysate 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.

Approximate number of cells/datapoint is indicated on the graph. The dotted line represents assay background. This assay can detect Phospho (Ser256) in approximately 2,500 cells/datapoint.