AlphaLISA SureFire Ultra Human and Mouse Total FOXO3A Detection Kit, 10,000 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.

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 then 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). FOXO3A Phospho (Ser253) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 2,000 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 FOXO3A (Ser253) with a modest increase in Total levels (2.7-fold).

NIH/3T3 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 starved for 2 hours and then treated with increasing concentrations of insulin prepared in serum free medium for 30 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). FOXO3A Phospho (Ser253) 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 FOXO3A (Ser253) while total levels did not increase significantly.

Knockout validation of FOXO3A Total assay

Total FOXO3A levels were assessed in HEK293 wild type (WT) and HEK293 FOXO3A knockout (KO) (Abcam, ab260857) cell lines. Cells were cultured to confluency in T75 flasks at 37°C, 5% CO₂. Cells were lysed in 2 mL of Lysis Buffer.

Lysates were serially diluted in Lysis Buffer and Total FOXO3A 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 FOXO3A protein was only detected in HEK293 WT cells, confirming assay specificity.

Expression of FOXO3A in various cell lines

Adherent cell lines were seeded in a 96-well plate (40,000 cells/well) and incubated overnight at 37°C, 5% CO₂. Cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm).

Suspension cell lines were seeded in a 96-well plate (400,000 cells/well) and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm).

FOXO3A levels were evaluated using the AlphaLISA SureFire Ultra assay. For the detection step, 10 µL of cell lysate (approximately 4,000 adherent cells and 40,000 suspension 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.

Total FOXO3A protein is expressed in a wide range of cell types, with particularly high expression in HEK293, RPMI 8226 and mouse cell line NIH/3T3.