| Assay Method Information | |
| | Biological Activity of Disclosed Compounds In Vitro |
| Description: | The ability of compounds to inhibit endogenous, cell-bound CD73 enzyme activity was demonstrated using SK-MEL-28 cells, which express CD73 on their surface. The day before the experiment, 5000 cells were plated per well in a 96-well plate. Cells were washed twice with 200 μL reaction buffer (20 mM HEPES, pH 7.4, 125 mM NaCl, 1 mM KCl, 2 mM MgCl2, 10 mM glucose) to remove residual inorganic phosphate. After washing, assays contained serial dilutions of test compounds and 100 μM of AMP in a total volume of 200 μL reaction buffer, with a final DMSO concentration ≤0.5%. After 30 minutes at room temperature, supernatant was removed from the cells. A volume of 100 μL Malachite Green (Cell Signaling Technology, Cat. No. 12776) was added to 25 μL of supernatant. After 5 minutes at room temperature, absorbance at 630 nm was determined on a microplate spectrophotometer. The concentration of inorganic phosphate was determined using a phosphate standard curve to determine IC50. |
| Affinity data for this assay | |
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