AlphaLISA SureFire Ultra Human and Mouse Total FOXO1 Detection Kit, 100 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.

Validation of FOXO1 Total in cells treated with insulin

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 change in Total levels.

Validation of FOXO1 Total in HUVEC cells treated with Razuprotafib

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 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.

Knockout validation of FOXO1 Total assay

Total FOXO1 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 Total 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.

Total FOXO1 was only detected in WT cells, confirming assay specificity.

Expression of FOXO1 in various cell lines

Adherent cells were grown to confluency in a T175 flask at 37°C, 5% CO₂, and were lysed with Lysis Buffer at a density of 0.5 x 10⁶ cells/mL. Suspension cells were harvested, washed in HBSS and lysed with Lysis Buffer at 1.6 x 10⁶ cells/mL.

FOXO1 levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of cell lysate 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.

Total FOXO1 protein is expressed in a wide range of cell types with high expression in Ramos, C2C12 and Hep G2 cell lines.

Assay sensitivity - cell lysate

Cell lysate was prepared from HEK293 cells seeded in T175 flasks and cultured to confluence and then lysed with 3 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysates were 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 FOXO1 expression in less than 1,250 cells/datapoint.