Colony Stimulating Factor 1 Receptor (CSF1R) is a receptor tyrosine kinase that binds CSF1 and IL-34 to regulate the survival, proliferation, and differentiation of mononuclear phagocytes. Upon ligand binding, CSF1R undergoes dimerization and autophosphorylation, activating PI3K/AKT, MAPK, and JAK/STAT pathways. CSF1R is essential for tissue macrophage homeostasis, bone remodeling, and immune responses. In the tumor microenvironment, CSF1R signaling promotes tumor-associated macrophages that support cancer progression through immunosuppression and metastasis. Loss-of-function mutations in CSF1R are linked to adult-onset leukoencephalopathy and other neurodegenerative disorders, highlighting its critical role in microglial maintenance and brain health. CSF1R inhibitors are being developed as cancer immunotherapies and treatments for macrophage-driven diseases.
The AlphaLISA SureFire Ultra Mouse Phospho-CSF1R (Tyr697) is a sandwich immunoassay for the quantitative detection of phospho-CSF1R (Tyr697) in cellular lysates, using Alpha Technology.
Formats:
The HV (high volume) kit contains reagents to run 100 wells in 96-well format, using a 60 μL reaction volume.
The 500-point kit contains enough reagents to run 500 wells in 384-well format, using a 20 μL reaction volume.
The 10,000-point kit contains enough reagents to run 10,000 wells in 384-well format, using a 20 μL reaction volume.
The 50,000-point kit contains enough reagents to run 50,000 wells in 384-well format, using a 20 μL reaction volume.
AlphaLISA SureFire Ultra kits are compatible with:
Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
AlphaLISA SureFire Ultra kits can be used for:
Cellular kinase assays
Receptor activation studies
High-throughput screening for preclinical studies
Phospho-AlphaLISA SureFire Ultra assay principle
The Phospho-AlphaLISA SureFire Ultra assay measures a target protein when phosphorylated at a specific residue in a biological sample (e.g. cell lysate).
The assay uses two antibodies which recognize the phospho epitope and a distal epitope on the target protein. AlphaLISA assays require two bead types: Acceptor and Donor Beads. Acceptor Beads are coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody, labeled with a CaptSure tag. Donor Beads are coated with streptavidin to capture one of the detection antibodies, which is biotinylated. In the presence of phosphorylated protein, the two antibodies bring the Donor and Acceptor Beads in close proximity whereby the singlet oxygen transfers energy to excite the Acceptor Bead, allowing for the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of phosphoprotein present in the sample.
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 enables cell 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 robust 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 (Tyr697) 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 Tyr697, 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 (Tyr697) 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 (Tyr697) 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-(Tyr697) 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-Tyr697 expression in less than 1,000 cells/datapoint.
