• No results found

The influences of the compounds on the NF- B pathway were studied using the Raw Blue Cells assay from the InvivoGen. Raw Blue cells, which are derived from the macrophages, stably express a

NF-B induced gene, secreting embryonic alkaline phosphatase (SEAP). The change in the NF- NF-B pathway was determined by measuring the concentration of secreted SEAP in the medium using Quanti-Blue. The measurements were performed following the provided protocols with some adjustments.

The cells were seeded in the 96 wells plate with ~100,000 cells per well in 200 µL Dulbecco's Modified Eagle Medium (DMEM) containing 0.1% (v/v) zeocin and the plate was incubate overnight at 37°C. For the blank, no cells were seeded. The medium was removed and replaced by 180 µL of fresh medium containing 3 µM of the modulator compounds 23a or 23d. After 6 hour incubation at 37°C, 20 µL of NF- B stimulant (IFN- ) was added. Empty medium was used as the control. The plate was incubated overnight at 37°C. 150 µL of the incubated medium were transferred into a flat-bottom 96 wells plate containing 50 µL Quati-blue suspension. The plate was incubated for another 6 hours at 37°C before the absorbance was measured using a ThermoMax microplate reader at 650 nm.

The experiments were performed in triplicate, and the presented results were the average of three independent experiments with the standard deviation.

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