| Assay Method Information | |
| | Phospho cFMS Activity |
| Description: | Reagents and consumables were purchased from Sigma Aldrich, Gibco LifeTechnologies, BD Biosciences, Perkin Elmer, R&D Systems, Cell Signaling, Thermo Scientific (Pierce) and Santa Cruz Biotechnology. HEK293 cells overexpressing human cFMS (HEK293/hFMS) were cultured in RPMI media in T225 flasks and split twice a week. For the experiment, the cells were trypsinized, counted and diluted with serum-free Megacell media (Sigma Cat #M3817) to 600,000 cells/ml (30,000 cells/well). A serial dilution of test compounds was prepared by the Echo 555 (LABCYTE) using Echo LDV Plates, Cat #LP-0200; and 500 nl of each compound concentration was added to 96-well BD Biocoat poly-d-lysine plate (BD Cat #356640) in DMSO (0.5% final). 50 μL/well MegaCell serum-free media was then added to cover compounds before adding cells at 50 μL/well cells (30,000/well). The plates were spun down for 1 minute at 1000 rpm and then incubated on benchtop for 15-30 minutes; the plates were moved to a CO2 incubator at 37° C. for overnight incubation. White 96-well Perkin Elmer OptiPlates (Cat #6005509) were pre-coated with 50 ng/well (100 μL/well) anti-cFMS/CSF-1R (C-20) (Santa Cruz Cat #sc-692) in PBS, sealed with a foil seal, spun down at 1000 rpm for one minute and incubated overnight at 4° C.On the following day, the pre-coated OptiPlates plates were blocked with 200 ul/well 1% BSA in 1× PBST (PBS with 0.1% Triton-X) at room temperature for 2-3 hours. In parallel, 100 μL/well 2× hCSF1 (final 150 ng/ml) (R&D Systems, Cat #216-MC-025/CF) (or media as a negative control) was added to the HEK293/hFMS cells (BD culture plates) incubated overnight with compounds. On every plate 100% response (with CSF1 treatment) and 0% response (without CSF1) control columns were used to calculate percent inhibition of tested compounds and a Z′ prime value. Plates were incubated at 37° C. for 10 minutes. Media/hCSF1 was aspirated off and cells were lysed with 100 ul/well pre-chilled lysis buffer made up with lysis buffer (Cell Signaling Cat #9803), protease/phosphatase inhibitors (Pierce Cat #78444), and PMSF (Sigma Cat #93482). Plates were shaken for 60 seconds; then, spun at 3200 rpm for 5 minutes at 4° C. and kept on ice. 90 ul of the lysate was transferred to the pre-coated/blocked OptiPlates. The plates were then spun at 1000 rpm for 60 seconds and incubated overnight at 4° C. sealed.The next day lysates were removed from the plates; and plates were washed with 300 μL/well of 1× PBS 6 times using the Biotek washer. The remaining PBS on the plates was tapped out. 90 μL/well of 1:10,000 anti-phospho-Eu (Tyr 100) (Perkin Elmer Cat #AD0159) in 1% BSA in PBST was added to the plates; and plates were incubated for one hour at room temperature sealed. After one hour, the antibody was removed and plates were washed 6 times with 300 μL/well of PBST using the Biotek washer. 90 μL/well enhancement solution (Perkin Elmer Cat #4001-0010) was added next and the plates were sealed and shaken for 5 minutes. The signal was read immediately on the Perkin Elmer Envision for time-resolved fluorescence with excitation at 320 nm and emission at 615 nm.The data were analyzed by Pipeline Pilot to calculate IC50 values. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |