| Assay Method Information | |
| | Assessment of MPO and TPO Activity Assay (IC50) |
| Description: | The experiments were performed in phosphate-buffered saline (PBS), pH7.4. A 10 mM luminol (A4695, Sigma Aldrich, St Louis, MO, USA) stock was prepared in distilled water and further diluted in PBS to a final concentration of 100 μM. H2O2 was prepared as 1 mM stock in PBS, yielding a final concentration of 50 μM after addition into the assay. The compounds were serially diluted in DMSO in a separate plate as a 100× stock solution, and MPO (purified from HL60 cells) and TPO (produced in insect cells, RSR Ltd, Cardiff, UK), were diluted to yield a final concentration of approximately 14 and 150 ng/mL, generating 5600 and 9300 light counts per seconds (LCPS) upon incubation with luminol. The experiment was run by pipetting 2 μL 100× stock solution of the compound and 200 μL diluted enzyme in PBS into wells in a 96-well Optiplate (6005290, Perkin Elmer/Thermo Fischer, Waltham, MS, USA), followed by addition of 10 μL H2O2 containing PBS. Chemiluminescence measurement (Perkin Elmer Wallac Microbeta Trilux 1450-029 (12-detector), Turkuu, Finland) was started directly and recorded after 2, 10 and 15 minutes. Chemiluminescence recorded after 15 minutes was used to calculate the IC50 values. |
| Affinity data for this assay | |
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