| Assay Method Information | |
| | Inhibition of C5a-C5aR Binding |
| Description: | U937 cells were originally obtained from the American Type Culture Collection (ATCC) and transfected with human C5a receptor (C5aR). U937/C5aR cells were cultured at 37° C., 5% CO2 in RPMI1640 (Gibco) supplemented with 10% FBS (Gibco) and 350 g/ml Geneticin (Gibco) and passaged every 3 days to maintain the density range from 1×105 to 2×106 cells/ml. Human C5a biotinylation was performed according to the procedure provided by manufacturer (Thermo Scientific, A39257). 10 mM solution of Sulfo-NHS-LC-Biotin was prepared by adding 180μl ultrapure H2O to the 1 mg vial immediately. 12 μl 10 mM biotin reagent was added to 200 g human C5a solution, gently pipetted for 3 seconds, and incubated on ice for 2 hours. Amicon ultra-0.5 centrifuge filter device (Millipore, UFC5003BK) was pre-rinsed with Milli-Q H2O, centrifuged at 14000 g for 5 min immediately before use. Up to 500 d sample (diluted by PBS) was added to the device and capped. The device was spinned at 14000 g for approximately 5 min. 250 μl PBS was added to the filter device, spinned at 14000 g for 5 min, and repeatedly washed 6 times. The filter device was separated from the microcentrifuge tube and placed upside down in a clean microcentrifuge tube, spinned for 2 min at 1000 g to transfer the concentrated sample from the device to the tube. U937/C5aR cells were collected and washed twice by PBS, cells were suspended in PBS+0.1% BSA buffer at the density of 3×106 cells/ml. 100 μl cell suspension was added to a 96 well microplate. 50 μl compound diluted in assay buffer and 50 μl biotinylated ligand human C5a (30 nM) were added to corresponding wells in order and the plate was incubated on ice for 120 min and then centrifuged at 1000 rpm for 3-5 min at 4° C. Supernatant was removed and cells were washed by pre-cold PBS twice. 100 μl FITC conjugated streptavidin was added to cells, incubated on ice for another 30 min, and then centrifuged at 1000 rpm for 3-5 min at 4° C. Supernatant was removed and cells were washed by pre-cold PBS twice. 150 μl PBS was added to suspend the cells and signals were detected by FACS (Beckman, Cytoflex). IC50 values were calculated by GraphPad Prism software. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |