AlphaLISA SureFire Ultra Mouse Phospho-CSF1R (Tyr706) 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.

M-CSF induces a rapid phosphorylation of CSF1R in RAW 264.7 cells

RAW 264.7 cells 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 treated with 200 ng/mL human M-CSF for the indicated time points.

After treatment cells were lysed with 80 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). CSF1R Phospho (Tyr706) levels were evaluated using the AlphaLISA SureFire Ultra assay. 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.

M-CSF triggered a rapid phosphorylation of Tyr706, peaking at 2 minutes and decreasing thereafter.

M-CSF induces CSF1R phosphorylation in a dose-dependent manner

RAW 264.7 cells 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 treated with increasing concentrations of human M-CSF for 5 minutes.

After treatment cells were lysed with 80 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). CSF1R Phospho (Tyr706) and ERK Total levels were evaluated using respective AlphaLISA SureFire Ultra 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, M-CSF triggered a dose-dependent increase in the levels of Phospho CSF1R (Tyr706) while Total ERK levels remained unchanged.

Assay sensitivity - cell lysate dilution

Cell lysate was prepared from RAW 264.7 cells cultured to confluency in a T175 flask, treated with 100 ng/mL human M-CSF for 5 minutes and lysed in 4 mL of Lysis Buffer for 10 minutes at RT with shaking.

Lysate was serially diluted in Lysis Buffer and CSF1R p-(Tyr706) levels were evaluated by AlphaLISA SureFire Ultra. 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 per datapoint is indicated. The dotted line represents assay background. This assay can detect CSF1R p-Tyr706 expression in less than 1,000 cells/datapoint.