| Assay Method Information | |
| | Calcium++ Mobilization Assay |
| Description: | U937 cells (ATCC CRL-1593.2) were cultivated in RPMI1640 medium supplemented with 10% fetal bovine serum in a standard cell culture incubator. The day before conducting the assay, Dibutyryl-cAMP (0.5 mM working concentration) was added to cell cultures. Next day, cells were spun and resuspended in RPMI 1640 to a concentration of 40,000 cells per 50 μl. 40,000 cells were plated in one well in a 96 well poly-D-lysine coated plate for two hours to allow cells to adhere. After cell adherence, cytoplasmic calcium++ indicator (FLIPR Calcium 6 Assay Kit, Molecular Devices) was added to each well and incubated for 75 minutes at 37° C. Test compounds were diluted using a robotic liquid handler. The tips of the robotic liquid handler were changed after each mixing step. Test compounds were added into cell cultures at various concentrations (0.01 nM to 100 μM) for 15 minutes at 37° C. The cell culture plates were then incubated at room temperature for 30 minutes before being placed into Flexstation-3 plate reader (Molecular Devices). The Flexstation-3 was programmed to add recombinant C5a protein at various concentrations (1 nM to 10 nM) to cell culture plates and to monitor the change of fluorescence intensity, which correlates with cytoplasmic calcium concentration. |
| Affinity data for this assay | |
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