AlphaLISA SureFire Ultra Human Phospho-CSF1R (Tyr561) Detection Kit, 100 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.

PBMCs were isolated from healthy donors and cultured for 7 days in complete DMEM containing 20 ng/mL M-CSF to differentiate them into macrophages. Macrophages were seeded in a 96-well plate (40,000 cells/well) in complete DMEM and incubated overnight at 37°C, 5% CO2. The cells were starved for 2 hours in HBSS + 0.1% BSA and then treated with 100 ng/mL M-CSF for the indicated time points.

After treatment cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). CSF1R Phospho (Tyr561) 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.

M-CSF triggered a rapid phosphorylation of Tyr561, peaking at 5 minutes and then decreasing thereafter. Total CSF1R levels decreased around 50% at 30 minutes suggesting degradation occurs post-phosphorylation.

THP-1 cells were seeded in a 96-well plate (200,000 cells/well) in HBSS + 0.1% BSA and starved for 2 hours at 37°C, 5% CO2. The cells were treated with 100 ng/mL M-CSF for the indicated time points.

After treatment, the cells were spun down and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). CSF1R Phospho (Tyr561) 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, M-CSF triggered a rapid phosphorylation of Tyr561 while Total CSF1R levels decreased 2-fold at 30 minutes.

BeWo cells were seeded in a 96-well plate (60,000 cells/well) and incubated overnight at 37°C, 5% CO2. The cells were starved for 2 hours in HBSS + 0.1% BSA and then treated with 100 ng/mL M-CSF for the indicated time points.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). CSF1R Phospho (Tyr561) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 6,000 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, M-CSF triggered a rapid phosphorylation of Tyr561 while Total CSF1R levels decreased 2-fold at 30 minutes.

THP-1 cells were seeded in a 96-well plate (200,000 cells/well) in HBSS + 0.1% BSA and starved for 2 hours at 37°C, 5% CO2. The cells were treated with increasing concentrations of M-CSF for 5 minutes.

After treatment, the cells were spun down and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). CSF1R Phospho (Tyr561) 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, M-CSF triggered a dose-dependent increase in the levels of Phospho CSF1R (Tyr561) while Total levels remained unchanged.

BeWo cells were seeded in a 96-well plate (60,000 cells/well) and incubated overnight at 37°C, 5% CO2. The cells were starved for 2 hours in HBSS + 0.1% BSA and then treated with increasing concentrations of M-CSF for 5 minutes.

After treatment, the cells were lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). CSF1R Phospho (Tyr561) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 6,000 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, M-CSF triggered a dose-dependent increase in the levels of Phospho CSF1R (Tyr561) while Total levels remained unchanged.

IL-34 induces CSF1R phosphorylation in a dose-dependent manner

THP-1 cells were seeded in a 96-well plate (200,000 cells/well) in HBSS + 0.1% BSA and starved for 2 hours at 37°C, 5% CO2. The cells were treated with increasing concentrations of IL-34 for 5 minutes.

After treatment, the cells were spun down and lysed with 100 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). CSF1R Phospho (Tyr561) 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, IL-34 triggered a dose-dependent increase in the levels of Phospho CSF1R (Tyr561) with no significant changes to the Total levels.