Synergy htx s1lfa

Effects on cellular viability were analyzed by a resazurin-based assay (PrestoBlue™; Thermofisher, A13261) and spectrophotometric detection on a Synergy HTX S1LFA plate reader (BioTek) according to the manufacturer's instructions.

Cells were treated and stimulated by either LPS (10 ng/mL) or interferon-gamma (IFN-γ, 25 ng/mL) for 24 hr. Cytokine secretions were analyzed in technical duplicates in appropriately diluted medium from treated cells by Duo ELISA kits (R&D technologies) for human CXCL10 (dy266), IL-8 (dy208), and TNF-α (dy210) according to the manufacturer's instructions on a Synergy HTX S1LFA plate reader (BioTek). GainData® [40 ] was used with a four-parameter logistic regression standard curve for the calculation of cytokine concentrations.

COX (ovine/human) inhibitor screening assay kit (Cayman Chemical, Ann Arbor, MI, USA) was used according to the manufacturer’s instructions for measuring COX-1 and COX-2 inhibition rates. Briefly, COX reactions were performed. Background tubes were prepared for COX-1 and COX-2 in boiling water for three minutes. The inactivated enzymes were used to generate the background values. Then, 160 µL reaction buffer, 10 µL of heme, and 10 µL of COX-1 or COX-2 were added to reaction tubes for COX-1 or COX-2 100% initial activity tubes and inhibitor tubes. Next, 10 µL of inhibitors was added to inhibitor tubes, whilst 10 µL of vehicle was added to 100% initial activity tubes and background tubes. Celecoxib and indomethacin were administered at 0.01–10 µM concentrations, whereas compound 2e was applied at 10–100 µM concentrations. Then, these tubes were incubated for 10 min at 37 °C. Reaction was initiated by adding 10 µL of arachidonic acid to all the reaction tubes, mixed, and incubated for two minutes at 37 °C. Then 30 µL of the saturated stannous chloride solution was added to each tube. The prostaglandins were quantified by ELISA. The plate was read at 405 nm in an ELISA reader (BioTek Instruments Synergy HTX S1 LFA).